PRAS40 and DEPTOR are mTOR binding proteins that affect cell metabolism. Under catabolic conditions such as sepsis and glucocorticoid excess, there is an increase in total DEPTOR protein and a reduction in phosphorylation of PRAS40, suggesting that these proteins may modulate the mTOR-mediated protein synthetic response under normal and diseased conditions. The hypothesis of the present study was that knock down (KD) of PRAS40 or DEPTOR in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. PRAS40 and DEPTOR KD was achieved using lentiviral particles containing shRNA to target the mouse PRAS40 and DEPTOR mRNA sequence, whereas control cells were transfected with a scrambled control shRNA. KD reduced PRAS40 and DEPTOR mRNA and protein content by 90%. PRAS40 KD did not result in increased phosphorylation of mTOR substrates or increased protein synthesis, whereas, DEPTOR KD increased both phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and protein synthesis. The responsiveness of PRAS40 and DEPTOR KD myocytes to anabolic (IGF-I) and catabolic (AICAR) stimuli was unaltered. Both PRAS40 and DEPTOR KD myoblasts were larger in diameter and exhibited an increased mean cell volume compared to scramble control. PRAS40 KD cells had decreased phosphorylation (S807/S811) of pRb protein. In contrast, DEPTOR KD cells had an increased phosphorylation (S807/S811) of pRb protein which is critical for the G ₁-S phase transition, coincident with an increased percentage of cells in the S phase. Neither PRAS40 nor DEPTOR KD altered myoblast apoptosis as evidenced by the lack of change for cleaved caspase-3. Although DEPTOR KD myoblasts did not alter autophagy as determined by a lack of change in the ratio of LC3BII/LC3BI, PRAS40 KD myoblasts had a reduced ratio of LC3BII/LC3BI. While PRAS40 KD delayed myotube formation concurrent with delayed proliferation, DEPTOR KD had the opposite effect on myogenesis and proliferation. Finally, while in vivo DEPTOR KD (∼50% reduction) by electroporation into the muscle of C57/BL6 mice did not alter weight or protein synthesis in the control muscle, it prevented atrophy produced by 3 days of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that PRAS40 and DEPTOR are important regulators of protein metabolism in myocytes and demonstrate that, while PRAS40 is required for normal myoblast growth and function, decreasing DEPTOR expression is sufficient to ameliorate the atrophic response produced by immobilization.