Branched-chain amino acids (BCAA) are essential amino acids that serve as essential substrates and important regulators in the synthesis of body proteins. Our primary objective was to investigate the effect of calorie restriction with endurance exercise and supplementation of branched-chain amino acids (BCAA; leucine, isoleucine &Valine) on fractional protein synthesis rate. Further mTOR signaling pathway (4E-BP1 Thr37/46 and S6K1 Thr389) was studied to understand the molecular mechanism, involved in the regulation of translation initiation in skeletal muscle. Thirty six rats were randomly divided into three groups- -Ad libitum with exercise (Ad+ Ex) as controls, Calorie restricted with exercise (CR+ Ex) and Calorie restricted with exercise and supplemented with branched chain amino acid (CR +Ex +BCAA). Fractional rates of protein synthesis and indexes of translation initiation were measured after endurance exercise (2 h post exercise). Muscle protein fractional synthesis rate was increased (P <0.05, P <0.01) in CR + Ex +BCAA supplemented group in comparison to control and CR group. Administration of BCAA during CR with exercise increased (P < 0.05) phosphorylation of PKB, mTOR, eukaryotic initiation factor (eIF) 4E-binding protein-1, 70 kDa ribosomal protein S6 kinase (S6K1) and ribosomal protein S6. BCAA also increased (P < 0.05) the phosphorylation of ribosomal protein S6 kinase in skeletal muscles. The results suggest that the BCAA supplementation under calorie restricted condition with endurance exercise acts as nutrient signal to stimulate protein synthesis in skeletal muscles of rats by activation of mTOR signaling pathway and its components including 4E-BP1 and S6K1.