Depression is a public disorder worldwide. Despite the widespread use of venlafaxine in the treatment of depression, it has been associated with the incidence of toxicities. Hence, the goal of the current investigation was to evaluate the mechanisms of venlafaxine-induced cell death in the model of the freshly isolated rat hepatocytes. Collagenase-perfused rat hepatocytes were treated with venlafaxine and other agents. Cell damage, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential decline, lysosomal damage, glutathione (GSH) level were analyzed. Moreover, rat liver mitochondria were isolated through differential centrifugation to assess respiratory chain functionality. Our results demonstrated that venlafaxine could induce ROS formation followed by lipid peroxidation, cellular GSH content depletion, elevated GSSG level, loss of lysosmal membrane integrity, MMP collapse and finally cell death in a concentration-dependent manner. N-acetyl cysteine, taurine and quercetine significantly decreased the aforementioned venlafaxine-induced cellular events. Also, radical scavenger (butylatedhydroxytoluene and α-tocopherol), CYP2E1 inhibitor (4-methylpyrazole), lysosomotropic agents (methylamine and chloroquine), ATP generators (L-gluthamine and fructose) and mitochondrial pore sealing agents (trifluoperazine and L-carnitine) considerably reduced cytotoxicity, ROS generation and lysosomal leakage following venlafaxine treatment. Mitochondrion dysfunction was concomitant with the blockade of the electron transfer complexes II and IV of the mitochondrial respiratory system. Therefore, our data indicate that venlafaxine induces oxidative stress towards hepatocytes and our findings provide evidence to propose that mitochondria and lysosomes are of the primary targets in venlafaxine-mediated cell damage.