Skeletal muscle plays a critical role in energy metabolism and physical performance, and mitochondrial function is essential for maintaining metabolic activity in muscle cells. Mealworm is a protein-rich source with a high protein content, and Tenebrio molitor protein hydrolysate (TMH) has been reported to contain elevated levels of branched-chain amino acids (BCAAs). BCAAs are known to regulate mitochondrial function through the AMPK/SIRT1 signaling in porcine skeletal muscle satellite cells. However, the potential role of TMH in regulating mitochondrial biogenesis in skeletal muscle cells has not yet been investigated. In this study, we examined the effects of TMH on mitochondrial biogenesis in C2C12 myotubes. TMH treatment enhanced the expression of PGC-1α, a key regulator of mitochondrial biogenesis, and subsequently increased the protein levels of NRF1 and TFAM. Furthermore, TMH enhanced the expression of MyHC, suggesting an improvement in the functional properties of differentiated myotubes. Notably, inhibition of AMPK or SIRT1 suppressed TMH-induced mitochondrial biogenesis–related gene expression. Collectively, these findings suggest that TMH promotes mitochondrial biogenesis in skeletal muscle cells through the AMPK/SIRT1 signaling pathway and may contribute to improved metabolic capacity in muscle.