The enhanced rates of protein synthesis overcoming to the protein degradation resulted in a net increase in cellular protein accumulation that leads to skeletal muscle growth and hypertrophy. It has been well documented that mammalian target of rapamycin (mTOR) plays an essential role for the regulation of protein synthesis and subsequent hypertrophy in skeletal muscle. The protein kinase C (PKC)-dependent regulation of mTOR activity and subsequent protein synthesis/cell growth has been suggested, however, the exact molecular mechanisms underlying this regulation are poorly defined in skeletal muscle. The purpose of this study was to determine the regulatory mechanism in PKC-dependent pathway leading to mTOR activation in skeletal muscle cells. C2C12 murine myoblasts were treated with serum/antibiotics free media for 120 min and then stimulated with PKC activator phorbol 12-myristate 13-acetate (PMA) for 20 min (100 nM in serum/antibiotics free media), co-incubated with/without mTOR-specific inhibitor rapamycin (50 nM) and PKC-specific inhibitor bisindolylmaleimide I (BIM-I) (10 μM). Phosphorylation of the p70 ribosomal S6 kinase 1 (S6K1) at both Thr389 and Ser421/Thr424 sites, which show the functional activity of mTOR signaling, were markedly increased (6.21 ± 0.44 fold increase in Thr389 and 12.53 ± 0.59 fold increase in Ser421/Thr424 compared to the control, p > 0.01) in response to the PMA treatment. Conversely, inhibition of PKC-dependent pathway by BIM-I treatment completely blocked PMA-induced activation of mTOR signaling. Phosphorylation of tuberous sclerosis complex 2 (TSC2) at S664 site (ERK-specific residue), an upstream regulator of mTOR, was also prevented by PKC inhibition. Overexpression of Ras homolog enriched in brain (Rheb), a downstream target of TSC2 and an mTORC1 activator, was sufficient to activate mTOR signaling. We also identified that, in the absence of Rheb with using siRNA gene knock down, PMA-induced activation of mTOR signaling was significantly prevented (67% decrease in S6K1-Thr389 phosphorylation and complete inhibition of rpS6-Ser240/244 phosphorylation compared to the negative control). These observations demonstrated that, in C2C12 myoblasts, the PKC-dependent activation of mTOR signaling is mediated through TSC2 phosphorylation and its downstream target Rheb. It was also confirmed that Rheb is essential regulator of mTOR activation and enhanced protein synthesis in skeletal muscle cells.