Bovine mammary epithelial cells were used to evaluate the effects of different levels of Arginine (Arg) on enzymes related to Arg metabolism. A series of seven Arg concentrations in the medium as treatments were T0 (0.00 mg L⁻¹) as control group, and T0.25 (69.50 mg L⁻¹), T0.5 (139.00 mg L⁻¹), T1 (278.00 mg L⁻¹), T2 (556.00 mg L⁻¹), T4 (1112.00 mg L⁻¹), and T8 (2224.00 mg L⁻¹) as experiment groups, respectively. The quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analysis showed that the nitric oxide concentration, the expressions of endothelial nitric oxide synthase in mRNA, and enzyme level were all increased in response to enhanced Arg doses such that the T8 was the greatest group (P < 0.05). Four-fold Arg concentration improved gene expression and synthesis of arginase which then deceased when excessive Arg was supplied (P < 0.05). The expressions of ornithine aminotransferase mRNA and enzyme in T1 and T2 groups were significantly greater than that in the other groups (P < 0.05). Two-fold Arg was the optimum level for ornithine decarboxylase gene expression and enzyme synthesis among all seven treatments (P < 0.05). These somewhat various effects of Arg concentrations on four kinds of enzymes in different Arg metabolic pathways suggest that Arg might participate in regulating bovine mammary physiological function with an optimum concentration by influencing the enzymes in related metabolic pathways.