--- - |2- Mechanical overloading can lead to skeletal muscle damage instead of remodeling. This is attributed to the excessive apoptosis of myoblasts, mechanism of which remains to be elucidated. The present study aimed to investigate the involvement of endoplasmic reticulum stress (ERS) and caspase‐12 in mediating the stretch‐induced apoptosis of myoblasts. Myoblast apoptosis was evaluated by Hoechst staining, DNA fragmentation assay, Annexin V binding, and propidium iodide staining, as well as caspase‐3 and poly‐ADP‐ribose polymerase 1 cleavage. First, our results showed that apoptosis was elevated in a time‐dependent manner when myoblasts were subjected to cyclic mechanical stretch (CMS) for 12, 24, and 36 hr. Concomitantly, CMS triggered the ERS and caspase‐12 cleavage; ERS inhibitor GSK 2606414 suppressed the CMS‐induced cleavage of caspase‐12 and myoblast apoptosis. Silencing caspase‐12 attenuated the apoptosis of myoblasts under CMS. Furthermore, CMS‐induced myoblast apoptosis was partially recovered by overexpressing wild‐type caspase‐12 in caspase‐12‐silenced myoblasts. In contrast, overexpressing mutant caspase‐12 (D94N), which cannot be cleaved into the active caspase‐12 fragments, failed to accomplish the same effect. Finally, C2C12 overexpressing truncated caspase‐12 segment (TC‐casp12‐D94), which starts from Asp94 and ends at Asn419, underwent apoptosis under both static and stretched conditions. Interestingly, C2C12 myoblasts seemed to be resistant to stretch‐induced apoptosis upon low‐serum‐induced differentiation. In conclusion, our study provided evidence that caspase‐12 cleavage at Asp94, induced by ERS under mechanical stimuli, is the key molecule in initiating the stretch‐triggered apoptosis of myoblasts. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9473-9487, December 2018.