Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.