Team Meat co-founder Edmund McMillen announced via Twitter that the 'Wrath of Lamb' expansion will land in "late May".
The Binding of Isaac was released for PC and Mac in September 2011, reaching the milestone of 450,000 sales in January this year.
Nintendo last month refused to stock the title on its 3DS eShop, with McMillen attributing the decision to its "questionable religious content".
The 'Wrath of Lamb' update, priced at £3, is described as a "mega expansion" for Binding of Isaac. It will increase the game's item count from 131 to 205, adding trinkets and new bonuses.
McMillen has since confirmed that he is in discussions with Sony about bringing the title to its platforms.
Watch a trailer for The Binding of Isaac below: