St. John is probably best known for being the voice of Duke Nukem in the Duke Nukem video game series from 1996 onwards, starting with Duke Nukem 3D with re-casting of Joe Siegler and Todd Replogle of Apogee Software. He stated in an interview that the voice of Duke Nukem was based upon Clint Eastwood's voice with a lower pitch. In an interview on zergwatch.com, Jon St. John was asked about sound clips of his performance being used in a Ventrilo Harassment video, and replied "Yes...I was made aware of it by Jeron (an audio Producer at 3DR) who suggested I check it out prior to recording a special Duke greeting to the 3DR crew just before the Duke Nukem Forever trailer was released. "I laughed my head off! That lady was out of her mind pissed off!" and after being asked if he thought Nukem was "just another gig" he went on to say "Just another gig? Are you kidding? Duke is KING! Can you think of any other video game hero with his attitude, destructive force or good looks? I don't think so. I love playing the part of Duke and I'm honored to be associated with such a pop icon."

He has provided the voice for the drill instructor Dwight T. Barnes as well as various other Marines in Half-Life: Opposing Force. He has done the voices for Big the Cat in Sonic Adventure and Sonic Heroes, Trash Man, General Warthog and other characters in Twisted Metal 4, E-102 Gamma in Sonic Battle, the announcer in Sonic Advance 3, E-123 Omega in Sonic Heroes, Bolt Logan in Chrome and Chrome: SpecForce and Agent Michael Ford in Conduit 2 He also dubs the voice of professor Simon and Archibald in Runaway 2. He also provided the narrator's voice in Forsaken. Jon also voices characters for rides and attractions at the LEGOLAND theme park in Carlsbad, CA. and at Dollywood theme park in Tennessee, he's the voice of BUZZ the Buzzard, an audio animatronic character who introduces riders to the Mystery Mine ride. He was also the crew chief in NASCAR Racing 2003 Season, NASCAR Racing 2002 Season, and NASCAR Racing 4. In 2011, he voiced for an exclusive Heroes of Newerth announcer pack and also a special sound pack for the TeamSpeak voice communication software. He is currently working on voicing for Dudebro: My Shit Is Fucked Up So I Got to Shoot/Slice You II: It's Straight-Up Dawg Time and Dota 2, as well as providing PvP voiceovers for Guild Wars 2.