Abstract Oxaliplatin (OXA), a third-generation platinum chemotherapeutic, is widely used against various malignancies but increasingly associated with male reproductive toxicity. The molecular mechanisms underlying germ cell dysfunction, however, remain unclear. In this study, we provide novel insights into the differential cellular responses of murine spermatogenic cell lines GC-1 (spg) and GC-2 (spd) to OXA exposure, focusing on key hallmarks of cytotoxicity, including metabolic disruption, oxidative stress, cell cycle perturbation, and apoptosis. Our findings reveal markedly higher susceptibility of GC-1 (spg) cells to OXA, characterized by significant reductions in viability and proliferation accompanied by G1 phase arrest and enhanced nuclear accumulation of phosphorylated p53, indicating activation of a DNA damage response. Both cell types exhibited elevated levels of intracellular ROS and distinct nuclear abnormalities, suggesting oxidative stress as a central mediator of cytotoxicity. Western blot analyses further confirmed the upregulation of intrinsic apoptosis-associated proteins, including Apaf-1, Bax, and cleaved-PARP, despite the absence of pronounced caspase-3 activity. Collectively, these results uncover cell type-specific vulnerabilities and multifactorial mechanisms of OXA-induced gonadotoxicity, providing insights for the development of protective strategies against chemotherapy-associated reproductive damage.