Shewanella oneidensis is a model species for aquatic ecosystems and plays an important role in bioremediation, biofuel cell manufacturing and biogeochemical cycling. S. oneidensis MR-1 is able to generate hydrogen sulfide from various sulfur species; however, its catalytic kinetics have not been determined. In this study, five in-frame deletion mutants of S. oneidensis were constructed and their H2S-producing activities were analyzed. SirA and PsrA were the two major contributors to H2S generation under anoxic cultivation, and the optimum SO32- concentration for sulfite respiration was approximately 0.8 mM, while the optimum S2O32- concentration for thiosulfate respiration was approximately 0.4 mM. Sulfite and thiosulfate were observed to interfere with each other during respiration, and a high concentration of sulfite or thiosulfate chelated extracellular free-iron but did not repress the expression of sirA or psrA. Nitrite and nitrate were two preferred electron acceptors during anaerobic respiration; however, under energy-insufficient conditions, S. oneidensis could utilize multiple electron acceptors simultaneously. Elucidiating the stoichiometry of H2S production in S. oneidensis would be helpful for the application of this species in bioremediation and biofuel cell manufacturing, and would help to characterize the ecophysiology of sulfur cycling.