Acrylamide (AA) is a common chemical, produced during food processing and widely used in various industries and laboratory processes. Thus, AA causes a significant risk for human and animal health. Recently published studies have suggested that reproductive toxicity of AA and glycidamide (GA) was mainly due to the oxidative stress which can lead to cell apoptosis. The present experiment was conducted to investigate the effect of oxidative stress on the apoptosis of mouse Leydig (TM3) and Sertoli (TM4) cells induced by AA and its metabolite GA. TM3 and TM4 cells were exposed to AA (10 µM and 1 mM) and GA (1 µM and 0.5 mM) for 24 h. Following the exposure time, the Leydig and Sertoli cells were evaluated for measurement of cell viability, lactate dehydrogenase activity, lipid peroxidation and hydrogen peroxide levels, apoptosis/necrosis rate, and mRNA expression levels of apoptotic genes (caspase3, Bcl-2, Bax, and p53). The present study showed that AA and GA exposure caused decrease in cell viability and increase in excessive oxidative stress and apoptosis in both cell types. In conclusion, our in vitro results demonstrate that oxidative stress probably plays a major role in AA- and GA-induced apoptosis of Leydig and Sertoli cells.