Nuclear factor kappa B (NFkappaB) has emerged as a crucial regulator of cell survival, playing important functions in cellular resistance to oxidants and chemotherapeutic agents. Recent studies showed that NFkappaB mediates cell survival through suppression of the accumulation of reactive species (RS) and a control of sustained activation of the Jun-N-terminal kinase (JNK) cascade. This work was undertaken in order to evaluate the role of NFkappaB in modulating the pro-oxidant effects of supplementation with vitamin A (retinol, ROH) in Sertoli cells, a major ROH physiological target. In this work, we reported that ROH treatment increases mitochondrial RS formation leading to a redox-dependent activation of NFkappaB. NFkappaB activation played a pivotal role in counteract RS accumulation in ROH-treated cells, since NFkappaB inhibition with DNA decoy oligonucleotides or pharmacological inhibitors (BAY-117082) potentiated ROH-induced RS accumulation and oxidative damage. In the presence of NFkappaB inhibition, ROH-induced oxidative stress promoted a prolonged activation of the JNK-activator protein 1 (AP-1) pathway and induced significant decreases in cell viability. Inhibition of JNK-AP-1 with decoy oligonucleotides to AP-1 or JNK inhibitor SP600125 prevented the decreases in cell viability. Antioxidants blocked the persistent JNK-AP-1 activation, cell oxidative damage, and the decreases in cell viability induced by NFkappaB inhibition. Finally, our data point superoxide dismutase (SOD)2 as a potential antioxidant factor involved in NFkappaB protective effects against ROH-induced oxidative stress. Taken together, data presented here show that NFkappaB mediates cellular resistance to the pro-oxidant effects of vitamin A by inhibiting RS accumulation and the persistent and redox-dependent activation of JNK-AP-1 cascade.