Co-op

Rebirth features 2 player local co-op on PC. The Afterbirth DLC increased this to 4 player local co-op. The PlayStation 4 version of the game is capable of both local and online multiplayer via PS4's "Share Play" feature. (The Playstation 4 version can also support up to 4 players.)

Co-op players can join in at any time as a random baby, taking a Red Heart container away from Isaac and giving it to the second player. Subsequent pressing of the join button will despawn the baby, giving Isaac the Red Heart container back.

Babies share Isaac's stats except for health and damage; babies deal half of Isaac's damage. Babies always have flight, but they cannot interact with objects like items and pickups, except for certain special babies like Magnet Baby and Isaac 2.

Joining as a co-op baby will bring up a menu that allows the player to pick from 5 special babies. Dying as one of the available special babies will remove it from the pool.

If you start a normal game and player 2-4 press Start while in the starting room, it will pop up a mini character selection wheel. If a player dies, they become a ghost baby that can only deal minimal damage and press buttons. Once the boss of a floor is killed, the dead player is reborn with all the items they once had, but with half a heart. When a boss is killed, the item pedestal will spawn as many items as there are players. All players share resources (coins, bombs, and keys) but each have their own hearts and items. Once all players die, it's game over.

Notes (Co-op Babies)

 * Soul Hearts will be given to Player 2 before normal hearts, and half hearts will be given before full hearts. If player 2 dies, they can rejoin the game at the cost of another Red Heart container, as a new random Baby.
 * The appearance of a majority of the co-op babies is solely cosmetic, but there are several "special" babies with beneficial effects. However, some special babies have detrimental effects.
 * The last shooting familiar Isaac picked up will follow Player 2 instead of Isaac.
 * If Isaac has more than 6 total hearts, including Soul Hearts and Black Hearts, Player 2 will take two hearts instead of one.
 * If Isaac has a maximum of 12 hearts, including Soul Hearts and Black Hearts, Player 2 will take three hearts instead of one or two.
 * The maximum amount of hearts Player 2 can have is three Red Hearts and one Soul Heart, using.
 * This limit increases by one Red Heart container, Soul Heart, or Black Heart when using Red Baby, Blue Baby, or Black Baby, respectively.
 * Because at least 1.5 hearts are required for co-op, Player 2 cannot join when playing as.
 * On the Nintendo Switch, you must restart the game after setting up the controllers or the join button will not work.

Strategy (True Co-op)

 * not only heals herself with, but also heals all other co-op players for a Half Red Heart. It's best to pair Magdalene up with characters who benefit or rely heavily on red health, such as , , or.
 * can pair well with, as Cain can take Lazarus' pill to have it be a guaranteed positive or neutral pill for Cain.
 * will not convert pills unless Cain holds them, so it can still be a negative pill if used by Lazarus.
 * 's reroll mechanic will apply in co-op, but this still includes the reroll of any items not labelled as "offensive".
 * Since there will be significantly less health-up sources as a result, it's recommended to pair with characters who can't use red health like, , , , , , or .  can also work since his Bag of Crafting entirely bypasses the reroll mechanic.
 * and will trigger for the player holding it regardless of which player killed the enemy.

Possessor
There is a hidden multiplayer game mode that allows a second player to control enemies and bosses in a way similar to Friend Finder item. To access it, the second player should hold both bumpers on their gamepad and press the Start/Options button (On keyboard, the second player should hold Q and E, then press Enter). After this, a message saying that a player has joined as the possessor will appear in the Debug Console. It works properly for most normal enemies and some specific bosses, such as Ultra Greed, but for the rest, controls are limited or do not work at all.