Geobacter sulfurreducens, an Fe(III)-reducing deltaproteobacterium found in anoxic subsurface environments, contains 4 NiFe hydrogenases. Hyb, a periplasmically oriented membrane-bound NiFe hydrogenase, is essential for hydrogen-dependent growth. The functions of the three other hydrogenases are unknown. We show here that the other periplasmically oriented membrane-bound NiFe hydrogenase, Hya, is necessary for growth after exposure to oxidative stress when hydrogen or a highly limiting concentration of acetate is the electron source. The beneficial impact of Hya on growth was dependent on the presence of H(2) in the atmosphere. Moreover, the Hya-deficient strain was more sensitive to the presence of superoxide or hydrogen peroxide. Hya was also required to safeguard Hyb hydrogen oxidation activity after exposure to O(2). Overexpression studies demonstrated that Hya was more resistant to oxidative stress than Hyb. Overexpression of Hya also resulted in the creation of a recombinant strain better fitted for exposure to oxidative stress than wild-type G. sulfurreducens. These results demonstrate that one of the physiological roles of the O(2)-resistant Hya is to participate in the oxidative stress defense of G. sulfurreducens.