During the peri-implantation period in sheep, L-arginine (L-Arg) in the uterine lumen is an essential substrate for the synthesis of nitric oxide (NO), by nitric oxide synthase (NOS), and polyamines, via arginase and ornithine decarboxylase, that are required for survival and development of ovine conceptuses (embryo and its extraembryonic membranes). L-Arginine can stimulate hypertrophy, hyperplasia, and differentiation of the ovine conceptus trophectoderm; however, the responsible signal transduction cascade has not been determined. Therefore, this study examined possible signaling pathways mediated by L-Arg, as well as the effects of two NO donors (S-nitroso-N-acetyl-DL-penicillamine and diethylenetriamine) and putrescine (precursor for spermidine and spermine) on oTr cell proliferation. Further, the inhibition of these effects by L-NAME (L-nitro-arginine methyl ester, an inhibitor of NOS) and nor-NOHA (N-omega-hydroxy-nor-arginine, an inhibitor of arginase) was assessed. L-Arginine treatment increased the abundance of phosphorylated MTOR, RPS6K, and EIF4EBP1 in oTr cells. Consistent with activation of these cell-signaling molecules, L-Arg increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of L-Arg were partially inhibited by both L-NAME and nor-NOHA. These results indicate that L-Arg enhances production of polyamines and NO and activates the MTOR/FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation and migration of oTr cells.