Amino acids, especially branched-chain amino acids such as l-Leucine, have been revealed to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through mTOR signaling pathway. In this study, we showed that a cationic amino acid, l-Arginine, also activated this signaling pathway in a rapamycin-sensitive manner in rat intestinal epithelial cells, and this l-Arginine-induced amino acid signal transduction involved the cationic amino acid transport system. The manner of l-Arginine- and l-Leucine-induced activation of p70 S6 kinase depended on the stimulation time and the concentration of each amino acid, which suggested that the mechanism of this amino acid signal acceptance might be saturable. l-Arginine and l-Leucine induced activation of p70 S6 kinase and phosphorylation of 4E-BP1 in a rapamycin-sensitive manner, which suggested the involvement of mTOR signaling pathway in these effects. l-Arginine-induced activation of p70 S6 kinase was inhibited by NG-Methyl-L-Arginine (NMMA) and L-N5-(1-Iminoethyl) Ornithine (NIO), inhibitors of nitric oxide synthase (NOS) which also block cationic amino acid transporters, system y(+). However, l-Leucine-induced activation of p70 S6 kinase was not affected with treatment of NOS inhibitors. In conclusion, l-Arginine regulates p70 S6 kinase activity and phosphorylation of 4E-BP1 through mTOR signaling pathway, which involves system y(+), cationic amino acid transporters.