Seafloor hydrothermal deposits form when hydrothermal fluid mixes with ambient seawater, and constituent sulfide minerals are usually interpreted to precipitate abiogenically. Recent research drilling at Izena Hole and Iheya North Knoll in the middle Okinawa Trough (East China Sea), combined with secondary ion mass spectrometry determinations of δ34S in pyrite grains, provides compelling evidence that the initial stage of subseafloor sulfide mineralization is closely associated with microbial sulfate reduction. During the sulfide maturation process, pyrite textures progress from framboidal to colloform to euhedral. Pyrite δ34S has highly negative values (as low as –38.9‰) in framboidal pyrite, which systematically increase toward positive values in colloform and euhedral pyrite. Sulfur isotope fractionation between seawater sulfate (+21.2‰) and framboidal pyrite (–38.9‰) is as great as –60‰, which can be attained only by microbial sulfate reduction in an open system. Because framboidal pyrite is commonly replaced by chalcopyrite, galena, and sphalerite, framboidal pyrite appears to function as the starting material (nucleus) of other sulfide minerals. We conclude that framboidal pyrite, containing microbially reduced sulfur, plays an important role at the initial stage of subseafloor sulfide mineralization.