Arginine, which is metabolized into ornithine, proline, and nitric oxide, plays an important role in embryonic development. The present study was conducted to investigate the molecular mechanism of arginine in proliferation, differentiation, and physiological function of porcine trophoblast cells (pTr2) through metabolic pathways. The results showed that arginine significantly increased cell viability (P<0.05). The addition of arginine had a quadratic tendency to increase the content of progesterone (P=0.06) and protein synthesis rate (P=0.03), in which the maximum protein synthesis rate was observed at 0.4 mM arginine. Arginine quadratically increased (P<0.05) the intracellular contents of spermine, spermidine and putrescine, as well as linearly increased (P<0.05) the intracellular content of NO in a dose-dependent manner. Arginine showed a quadratic tendency to increase the content of putrescine (P=0.07) and a linear tendency to increase NO content (P=0.09) in cell supernatant. Moreover, increasing arginine activated (P<0.05) the mRNA expressions for ARG, ODC, iNOS and PCNA. Furthermore, inhibitors of arginine metabolism (L-NMMA and DFMO) both inhibited cell proliferation, while addition of its metabolites (NO and putrescine) promoted the cell proliferation and cell cycle, the mRNA expressions of PCNA, EGF and IGF-I, and increased (P<0.05) cellular protein synthesis rate, as well as estradiol and hCG secretion (P<0.05).