Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant that has been used as an additive for fat- or oil-containing foods. The exposure index value increases with extended usage of the chemical. Further, estimated total amount of BHT could exceed standard regulation, considering dietary intake or another exposure. Although BHT may induce side effects in reproductive systems, adequate research had not yet been performed to confirm them. In this study, we investigated the effects of BHT on mouse Leydig cells (TM3), which are components of testis. Our results indicated that BHT suppressed cellular proliferation and induced cell cycle arrest in TM3 cells. Moreover, BHT hampered cytosolic and mitochondrial calcium homeostasis in TM3 cells. Furthermore, BHT treatment led to endoplasmic reticulum (ER) stress and DNA fragmentation, simultaneously stimulating intrinsic apoptosis signal transduction. To elucidate the mode of action of BHT on Leydig cells, we performed western blot analysis and confirmed the activation of the PI3K/AKT and MAPK pathways. Collectively, our results demonstrated that BHT has toxic effects on mouse Leydig cells via induction of calcium dysregulation and ER-mitochondria dysfunction.