TCDD, as an endocrine disruptor, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underlying the testicular effects of TCDD, the potential toxicity of TCDD on Sertoli cells was investigated. Furthermore, the study aims to delineate whether mitochondrial fractions of Sertoli cells are involved in mediating the testicular effects of TCDD. Adult rat Sertoli cells were incubated with (5, 10 or 15nM) of TCDD for 6, 12 or 24h. Cell viability, lactate and LDH leakage into media along with lipid peroxidation, ROS generation, SOD, CAT, GPx, GR, γ-GT and β-glucuronidase activities, GSH content and Δψ m were measured. Superoxide anion production, COX and cardiolipin content were measured in mitochondrial fractions. Cell viability was significantly decreased while lactate and LDH leakage into media were increased. ROS generation along with lipid peroxidation was also increased. SOD, CAT, GPx, GR activities and GSH content were significantly decreased. γ-GT and β-glucuronidase activities were also decreased. Superoxide anion production was increased while COX activity and cardiolipin content were decreased in mitochondrial fractions. Moreover, the Δψ m was significantly decreased as measured in Sertoli cells. In conclusion, TCDD impairs Sertoli cell functions and this effect is, at least in part, attributed to oxidative stress. We have also found that TCDD increases mitochondrial superoxide anion production and decreases Δψ m , COX activity and mitochondrial cardiolipin content. Our findings suggest that mitochondria may play an important role in ROS production, leading to the TCDD-induced oxidative stress response and resulting toxicological consequences in rat Sertoli cells.