Male hypogonadism associates with type 2 diabetes, and T can protect pancreatic β-cells from glucotoxicity. However, the protective mechanism is still unclear. This study thus aims to examine the antiapoptotic mechanism of T in pancreatic β cells cultured in high-glucose medium. T (0.0005-2 μg/mL) was added to INS-1 cells cultured in basal glucose or high-glucose media. Then cellular apoptosis, oxidative stress, and cell viability were measured. Endoplasmic reticulum (ER) stress markers and sensors and the antiapoptotic protein (B-cell lymphoma 2) were investigated by real-time PCR and Western blot analysis. ER stress markers were also measured in male mouse pancreatic islet cultured in similar conditions. T (0.05 and 0.5 μg/mL) did not have any effect on apoptosis and viability of INS-1 cells cultured in basal glucose medium, but it could reduce apoptosis and increase viability of INS-1 cells cultured in high-glucose medium. The protective effect of T is diminished by androgen receptor inhibitor. T (0.05 μg/mL) could significantly reduce nitrotyrosine levels, mRNA, and protein levels of the ER stress markers and sensor those that were induced when INS-1 cells were cultured in high-glucose medium. It could also significantly increase the survival proteins, sarco/endoplasmic reticulum Ca(2+) ATPase-2, and B-cell lymphoma 2 in INS-1 cells cultured in the same conditions. Similarly, it could reduce ER stress markers and increase sarco/endoplasmic reticulum Ca(2+) ATPase protein levels in male mouse pancreatic islets cultured in high-glucose medium. T can protect against male pancreatic β-cell apoptosis from glucotoxicity via the reduction of both oxidative stress and ER stress.