The biological and chemical potential for electron shuttling via humic acids was evaluated by analyzing the depth distribution of humic-acid-reducing and iron-reducing bacteria in a freshwater sediment, and correlating it to the redox characteristics of humic acids and iron. Physicochemical analysis of profundal sediments of Lake Constance revealed a distinct stratification, with oxygen respiration, microbial iron and sulfate reduction, and methanogenesis allocatable to defined layers. Among the acid-extractable iron in the surface layer, ferric iron (Fe(III)) was dominant, whereas ferrous iron (Fe(II)) prevailed below 2 cm depth. Humic acids showed a higher electron-accepting (oxidizing) capacity in the surface layer and a higher reducing capacity in deeper layers. The more reduced redox state of humic acids in deeper layers was probably due to reduction by humic-acid-reducing microorganisms. Most-probable-number analysis revealed that the sediments contained populations of humic-acid-reducing bacteria that (i) were substantially larger than those of the iron-reducing bacteria in the respective sediment layers and (ii) were in the same range as those of the fermenting bacteria. Our results suggest that microbial reduction of humic acids and subsequent chemical reduction of poorly soluble iron(III) minerals by the reduced humic acids represents an important path of electron flow in anoxic natural environments such as freshwater sediments.