Retinol (vitamin A) supplementation induced oxidative stress and mitochondrial reactive oxygen species (ROS) overproduction in cultured Sertoli cells. Our group demonstrated that ROS overproduction induced by retinol was the first step to abnormal Sertoli cells proliferation, pre neoplasic transformation or apoptose. Cytoskeleton architecture is said to have a central role in all these phenomena so during the last year  our group decided to investigate  the effects of retinol treatment on cytoskeleton in cultived Sertoli cells. We observed that cytoskeleton proteins showed decrease on oxidative damage index and morphology modifications. In this work we investigate the role of F-actin integrity on the ROS production in retinol treated Sertoli cells co-treated or not with Cytochalin B (CB), a F-actin disruption drug. We quantified superoxide production on isolated Sertoli cells sub-mitochondrials particles and also in tottal cellular enviroment by tert-butyl hydroperoxide chemiluminescent assay. We also mensured the mitochondrial membrane potential in cells that received retinol co-treated or not with CB to correlate actin cytoskeleton and mitochondrial ROS production. CB is said to have a secondary effect blocking glucose transport, so we quantified glucose concentration in Sertoli cell at the end of treatment time and no significat differences were found. Our results suggested that co-treatment with CB was able to revert ROS overproduction induced by retinol, this revertion occur also in isolated mitochondrial as in tottal cellular enviroment. We also find that co-treatment with CB modulated the mitochondrial membrane potential induced by retinol. All this finds sugest that actin microfilaments architecture is essential to ROS over-production induced by retinol treatment in Sertoli cells, and actin modifications can modulate mitochondrial events like the membrane potencial. Since actin participation has an increase importance in oxidative stress events, we believe that this work helps to clarify the correlation between ROS overproduction and cytoskeleton proteins in retinol treated Sertoli cells.