Cypermethrin, one kind of pyrethroid pesticides, has been shown to act as endocrine-disrupting chemicals (EDCs). The purpose of this study was to explore the roles of Sertoli cell apoptosis through mitochondrial pathway associated with calcium (Ca²⁺) in cypermethrin-induced male reproductive toxicology. The mouse Sertoli cells TM4 were cultured with 0 μM, 10 μM, 20 μM, 40 μM and 80 μM of cypermethrin. We used flow cytometry, Fluo-4 AM, western blot and JC-1 Assay Kit to examine apoptosis, intracellular Ca²⁺, expressions of mitochondrial apoptotic pathway-related proteins and mitochondrial membrane potential. We found cypermethrin increased apoptosis rate of TM4 cells significantly and with a significant increase in intracellular Ca²⁺ concentration. Cypermethrin significantly decreased the protein expressions of cytosolic B-cell lymphoma-2 (Bcl-2) and mitochondrial cytochrome c (Cyt-c). The protein expressions of cytosolic Bcl-2-associated x (Bax), Cyt-c, cleaved caspase-3, calmodulin (CaM), Ca²⁺/CaM-dependent protein kinases II (CaMKII) and phosphorylated CaMKII were increased significantly in cypermethrin-exposed TM4 cells. Cypermethrin decreased mitochondrial membrane potential significantly. Then, Bcl-2 family and Ca²⁺/CaM/CaMKII pathway participate in cypermethrin-induced homeostasis. Ca²⁺ overload activates mitochondrial pathway by increasing permeability of mitochondrial membrane and decreasing mitochondrial membrane potential. We suggest cypermethrin induces Sertoli cell apoptosis involving mitochondrial pathway associated with Ca²⁺ regulated by Bcl-2 family and Ca²⁺/CaM/CaMKII pathway. The study provides a new insight into mechanisms involved in cypermethrin-induced male reproductive toxicology.