The potential of sulfate-reducing bacteria (SRB) as biocatalysts for H-2 production from formate was recently demonstrated, but the electron transfer pathways involved were not described. In the present work, we analyzed the H-2 production capacity of five Desulfovibrio strains: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio fructosivorans, and Desulfovibrio gigas. D. vulgaris showed the highest H-2 productivity (865 mL L-medium (-1)), and D. gigas the lowest one (374 mL L-medium (-1) of H-2). The electron transfer pathways involved in formate-driven H-2 production by these two organisms were further investigated through the study of deletion mutants of hydrogenases (Hases) and formate dehydrogenases (Fdhs). In D. vulgaris, the periplasmic FdhAB is the key enzyme for formate oxidation and two pathways are apparently involved in the production of H-2 from formate: a direct one only involving periplasmic enzymes and a second one that involves transmembrane electron transfer and may allow energy conservation. In the presence of selenium, the Hys [NiFeSe] Hase is the main periplasmic enzyme responsible for H-2 production, and the cytoplasmic Coo Hase is apparently involved in the ability of D. vulgaris to grow by converting formate to H-2, in sparging conditions. Contrary to D. vulgaris, H-2 production in D. gigas occurs exclusively by the direct periplasmic route and does not involve the single cytoplasmic Hase, Ech. This is the first report of the metabolic pathways involved in formate metabolism in the absence of sulfate in SRB, revealing that the electron transfer pathways are species-specific.