[Objective] To investigate the effects of different concentrations of amino acids on urea cycle in primary hepatocytes isolated from piglet, and explore the potential mechanism.[Method] Primary hepatocytes isolated from 5-day old piglet were cultured in medium containing different concentrations of amino acids (AA)(1, 2, or 4 times of physiological concentration in serum). After 24 h, the supernatant and cells were collected. Urea and ammonia concentrations in the supernatant, glutaminase(GLS) and glutamine synthetase(GS) activities and ammonia concentration in cells were examined by colorimetry. The mRNA expression of genes involved in urea cycle, including carbamyl phosphate synthetase 1(CPS1), Argininosuccinate synthetase(ASS), Argininosuccinate lyase(ASL), Omithine transcarbamylase (OTC), Arginase (ARG) were detected by qRT-PCR.[Result] Urea and ammonia concentrations in the supernatant, and GLS activity in cells were significantly elevated in 4×AA group. Meanwhile, 4×AA remarkably increased the mRNA expression of CPS1, ASS and ASL genes in hepatocytes. NH₄Cl at 1.0 and 2.0 mmol·L^–1 concentrations significantly promoted urea synthesis in the cell.[Conclusion] High concentration of AA might accelerate ammonia accumulation by GLS and enhance the expression of the urea cycle enzymes (CPS1, ASS and ASL), which contribute to increased production of urea.