- Wentworth Miller as Michael Scofield (Season 1–4): Michael is Lincoln's brother and worked as a structural engineer before devoting full-time to his brother's case. In order to save his brother's life, Michael creates an elaborate plan to help his brother escape from prison. In an interview, Paul Scheuring recalled that most of the actors who tested for the role "would come in playing mysterious, but it was so cheesy and false." A week before the start of production, Miller auditioned for the role and impressed Scheuring with his performance; he was cast the following day.
- Dominic Purcell as Lincoln Burrows (Season 1–4): Lincoln is a high school drop-out and a convicted felon, who is wrongfully accused of and charged with the murder of Terence Steadman, the brother of the Vice President of the United States. Purcell was cast three days before the start of production and consequently, he was the last actor to join the original cast. He auditioned for the role while he had a recurring role as Tommy Ravetto on North Shore. Since working on John Doe, Purcell has had an amiable relationship with Fox. Hence, he was sent the pilot script of Prison Break. Scheuring's first impression of Purcell did not convince him as a fit for the role since the actor went to the audition with his hair styled and a tan. However, Purcell's acting won the role. He arrived on the set on the first day of filming with a shaved head, which amazed Scheuring with the physical likeness of the series' two leading actors.
- Robin Tunney as Veronica Donovan (Season 1–2): Veronica is Michael and Lincoln's childhood friend and decides to review Lincoln's case at Michael's insistence. She becomes Lincoln's lawyer and appears as a major character in the first season.
- Marshall Allman as Lincoln "L. J." Burrows Jr. (Season 1–4): L. J. is the teenage son of Lincoln Burrows and is greatly affected by his father's death sentence. He is forced into hiding after he becomes the target of the people who want Lincoln dead.
- Amaury Nolasco as Fernando Sucre (Season 1–4): Sucre develops a friendship with Michael during time at Fox River State Penitentiary, where he was his cell-mate. He becomes Michael and Lincoln's ally, and provides comic relief to the show. His character's story focuses mainly on his wish to reunite with his girlfriend. Upon receiving the pilot script, Nolasco's first thought was that it was "one of those failed pilots that the network did not really want" since most of the series pilots would have started production by that time. Admitting that he does not like to read, Nolasco was amazed that the script was a "huge page-turner". Prior to his last audition for the role, Nolasco recalled his nervousness, which grew when Paul Scheuring told him that he was their favourite choice. Subsequently, he was cast in the role.
- Robert Knepper as Theodore "T-Bag" Bagwell (Season 1–4): T-Bag appears in all four seasons of the series as a cunning, violent and manipulative psychopath, consistently underestimated by those around him. T-Bag will stop at nothing to get what he wants and lets nothing stand in his way.
- Peter Stormare as John Abruzzi (Season 1-2): Due to his role as the leader of a Chicago mafia, Abruzzi became a prominent figure at Fox River State Penitentiary. He agrees to provide an escape plane for Michael in exchange for the location of the eyewitness to his crimes, Otto Fibonacci. He appears regularly in the first half of the first season and makes selected appearances towards the end of the first season and the beginning of the second season.
- Rockmond Dunbar as Benjamin Miles "C-Note" Franklin (Season 1–2, 4): Desperate for his family, C-Note blackmails Michael at Fox River to join his escape team. He appears in the series as a major character in the first and second seasons.
- Wade Williams as Brad Bellick (Season 1–4): Appearing in all four seasons, Bellick was introduced as the captain of Fox River's correctional officers. After reading the pilot script, Williams initially did not want to portray the role of Bellick because the character was "horrible and despicable". His reluctance stemmed from being the father of a four-year-old daughter. However, his manager persuaded him to audition for the role and Williams landed the role of Bellick.
- Sarah Wayne Callies as Sara Tancredi (Season 1–2, 4): Sara is the prison doctor at Fox River and the daughter of Governor Frank Tancredi, who is linked into the plot that brings Lincoln to Fox River. She takes a liking to Michael and eventually aids his escape. She ultimately joins them on the run. Callies was the first actress the producers saw at the audition for the role of Sara Tancredi and was also the first to become a principal cast member.[30][32]
- Paul Adelstein as Paul Kellerman (Season 1–2, 4): Kellerman was introduced as a Secret Service agent working for the Vice President to make sure that the execution of Lincoln Burrows goes smoothly. Eventually, his character changes from that of a villain to an ally to Michael and Lincoln. He appears as a major character in the first and second seasons.
- William Fichtner as Alexander Mahone (Season 2–4): Introduced as an FBI agent in the second season, Mahone's assignment was to locate the fugitives. Mahone is intellectually matched with Michael and his background unfolds as the series progresses. In season 3 he finds himself incarcerated with Michael in Sona and is eventually forced to become his ally through season 4.
- Chris Vance as James Whistler (Season 3-4): Whistler is incarcerated in Sona for the murder of the Mayor's son and appears as a major character in the third season. He also stars in the first episode of Season 4.
- Robert Wisdom as Norman "Lechero" St. John (Season 3): Appearing as a major character in the third season, Lechero is a prisoner at Sona who rules the prison as a dictator and a Panamanian drug kingpin. And was killed by T-bag at the end of season 3
- Danay Garcia as Sofia Lugo (Season 3-4): Sofia was introduced in the third season as Whistler's girlfriend, at the beginning of season 4 it is revealed she has started to date Lincoln Burrows.
- Jodi Lyn O'Keefe as Gretchen Morgan (Season 3–4): Introduced as "Susan B. Anthony", Gretchen is an operative for the company who is in charge of ensuring the escape of James Whistler.
- Michael Rapaport as Donald Self (Season 4): Introduced in season 4, Self is a Department of Homeland Security special agent who teams up with the gang to take down The Company.
