The main purpose of this work was to evaluate the effects of jugular L-arginine infusion on antioxidant mechanisms in lactating dairy cows challenged intravenously with lipopolysaccharide (LPS). Eight multiparous Holstein cows (609 ± 32 kg) at mid-lactation were randomly assigned to 5-d jugular infusions of Control (saline), Arginine (Arg, 18 g/d), LPS (0.2 μg/kg BW per d), and LPS + Arginine (0.2 μg/kg BW per d of LPS and 18 g/d of Arg) in a replicated 4 4 Latin square design with 4 infusion periods separated by 10-d. Jugular solutions of saline, Arg, LPS, and LPS + Arg were continuously infused using peristaltic pumps for approximately 6 h/d. Jugular vein serum samples were obtained on the last day of each infusion period before infusion (0 h) and at 3 and 6 h post-infusion. Compared with LPS treatment, Arg infusion increased the total antioxidant capacity and activity of glutathione peroxidase, but decreased malonaldehyde concentration (P < 0.05). The concentration of nitric oxide in serum and the activity of nitric oxide synthase were greater in LPS treatment compared with saline and Arg (P < 0.05). The Arg treatment significantly increased the serum insulin concentration at 3 h post-infusion compared with the saline treatment (P < 0.05), and that of LPS and LPS + Arg treatments were in between Arg and LPS treatments. No treatment effect was observed on the activities of superoxide dismutase and catalase (P > 0.05). In conclusion, enhancing the supply of Arg during an inflammatory challenge enhances antioxidant mechanisms in lactating dairy cows.