Isaac is the main character of the series, reappearing from the original Binding of Isaac. Isaac is the first playable character, being unlocked at the start of the game. Isaac starts with three red heart containers and one bomb by default. He will also start with The D6Allows Isaac to re-roll collectible items in the current room into other items, depending on the current room's Item Pool. once it is unlocked.
- The The D6Allows Isaac to re-roll collectible items in the current room into other items, depending on the current room's Item Pool. that Isaac starts with does not increase the special counter, as Isaac starts with it.
- Isaac is a reference to the biblical story of the binding of Isaac, in which Abraham is commanded by God to sacrifice his only son, Isaac, to prove his loyalty and love.
- "Then God said, 'Take your son, your only son, whom you love — Isaac — and go to the region of Moriah. Sacrifice him there as a burnt offering on a mountain I will show you.'" (Genesis 22:2)
- In a response to TBoI Youtuber Sinvicta of whether there was any concept art of what Isaac's original design was, Edmund McMillen confirmed that Isaac's design was originally one of an Octorock while showing very similar features of how the gameplay would be modeled by posting an old concept art.
- Edmund has stated that Isaac is left-handed, but in the opening cutscene, Isaac is holding a pencil with his right hand.
- This could be a reference to the Catholic bias against left-handedness, stemming from Matthew 25:33 proclaiming that God will put those deserving to ascend to Heaven on his right-hand side and put those deserving to descend to Hell on his left-hand side. Isaac's mother could have forced him to learn things the "righty" way for fear of him being "evil."
- Contrary to previous theories made about Isaac, he does not have a sister.
- Isaac is bald because his mother shaved his head.
- Isaac has Irritable Bowel Syndrome. 
Original concept art for a character who would eventually be remodeled to Isaac.