How bacteria catalyze membrane fission during growth and differentiation is an outstanding question in prokaryotic cell biology. Here, we describe a protein (FisB, for fission protein B) that mediates membrane fission during the morphological process of spore formation in Bacillus subtilis. Sporulating cells divide asymmetrically, generating a large mother cell and smaller forespore. After division, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Membrane fission releases the forespore into the mother cell cytoplasm. Cells lacking FisB are severely and specifically impaired in the fission reaction. Moreover, GFP-FisB forms dynamic foci that become immobilized at the site of fission. Purified FisB catalyzes lipid mixing in vitro and is only required in one of the fusing membranes, suggesting that FisB–lipid interactions drive membrane remodeling. Consistent with this idea, the extracytoplasmic domain of FisB binds with remarkable specificity to cardiolipin, a lipid enriched in the engulfing membranes and regions of negative curvature. We propose that membrane topology at the final stage of engulfment and FisB–cardiolipin interactions ensure that the mother cell membranes are severed at the right time and place. The unique properties of FisB set it apart from the known fission machineries in eukaryotes, suggesting that it represents a new class of fission proteins.