Amino acids are potent regulators of muscle protein synthesis and breakdown and have received considerable attention for the treatment of muscle wasting conditions. Arginine is critically involved in numerous physiological functions including providing substrate for the production of creatine, urea and nitric oxide (NO) and in the synthesis of new proteins. However, little is known about the direct effects of arginine on skeletal muscle protein synthesis during catabolic conditions. The aims of this study were to determine whether exogenous arginine could protect skeletal muscle cells from wasting directly and whether this effect was dependent on production of NO and/or activation of the rapamycin-sensitive mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. To explore these aims, we deprived mature C2C12 myotubes from nutrients and growth factors by incubating them in HEPES buffered saline with arginine or equimolar concentrations of alanine (control). Our results show that arginine: increased the ratio of phosphorylated to total mTOR (146 %), S6 (40 %) and 4EBP1 (69 %); increased protein synthesis (69 %) during the first hour of treatment; and increased myotube diameter by ~15 %. Experiments using the NO synthase inhibitor L-NG-Nitroarginine Methyl Ester showed a NO-independent protection from muscle wasting. On the other hand, the mTORC1 inhibitor rapamycin prevented increases in phosphorylated S6, protein synthesis and myotube diameter. The activation of mTORC1 and protein synthesis by arginine was not associated with changes in the phosphorylation status of Akt, but rather increased the expression of the amino acid-sensitive type III PI3-kinase Vps34 signalling protein. These data support a direct role for arginine in the regulation of mTORC1 in skeletal muscle.