Although pyrite is the main sedimentary form of sulfur, an ample mechanistic comprehension of its formation in low-sulfate environments is lacking. Applying high depth-resolution multigeochemical and stable sulfur isotope composition (δS) analysis of a sediment core recovered from a large shallow freshwater lake (Baiyangdian) in north China, we show that the pyrite forms dominantly in the top 4 cm layer and the participating sulfide stems primarily from mineralization of reduced organic sulfur in biomass. This mechanism was further verified by the formation of pyrite in anoxic incubation of biomass ( L. or ) with hematite in the absence of external sulfate. This finding reveals an alternative pathway other than microbial sulfate reduction (MSR) for producing sulfide to form pyrite in low-sulfate sediments.