Spermatogonial stem cells (SSCs) represent a unique population of germ cells with self-renewal potential. Although reactive oxygen species (ROS) are considered toxic to germ cells, we recently showed that moderate levels of ROS are required for SSC self-renewal and that Nox1 is involved in ROS generation. In this study, we showed that self-renewal factor treatment induces Nox3 to trigger SSC self-renewal. Nox3 was transiently expressed in cultured spermatogonia by FGF2 and GDNF stimulation, whereas Nox1 was expressed predominantly during the stable phase of proliferation. Nox3 inhibition by short hairpin RNA reduced cytokine-induced ROS generation and limited the proliferation of cultured spermatogonia. Although Nox3 overexpression revealed no apparent effect, depletion of Nox3 decreased the number of SSCs in both cultured spermatogonia and freshly isolated testis cells. Our results suggest that self-renewal of SSCs is regulated by sequential activation of different Nox genes, and underscore the complexity of ROS regulation in the self-renewal division of SSCs.