This study evaluated the effects of arginine (Arg) or N-carbamylglutamate (NCG) on inflammation, antioxidant property, and antioxidant-related gene expression in rat spleen under oxidative stress. A total of 52 rats were randomly distributed into 4 treatment groups with 13 replicates per group. Rats were fed a basal diet (BD) or BD supplemented with Arg or NCG for 30 days. On day 28, half of the BD-fed rats were intraperitoneally injected with sterile saline (control group), and the other half with 12 mg/kg body weight of diquat (DT; DT group). The other 2 diet groups were intraperitoneally injected with 12 mg/kg body weight of DT with either Arg (1%) (DT + Arg) or NCG (0.1%) (DT + NCG). Rat spleen samples were collected for analysis at 48 h after DT injection. Results showed that DT damaged the antioxidant defense in rats compared with the control group (P < 0.05). Compared with the DT group, the DT + Arg and DT + NCG groups manifested improved anti-hydroxyl radical, catalase, and total superoxide dismutase (T-SOD) activities, increased glutathione content (P < 0.05), and decreased malondialdehyde content (P < 0.05). Moreover, compared with the DT group, the DT + Arg and DT + NCG groups enhanced mRNA expression of superoxide dismutase (SOD), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1(Keap-1), and mammalian target of rapamycin (mTOR) (P < 0.05). Both NCG and Arg significantly increased anti-inflammatory cytokine mRNA level but suppressed the pro-inflammatory cytokine mRNA expression under oxidative stress (P < 0.05). In summary, NCG and Arg effectively alleviated oxidative stress, improved the antioxidant capacity and regulated the antioxidant-related signaling molecular expression in rat spleen. N-carbamylglutamate and Arg reduced the inflammation in the spleen by mediating the gene expression of anti-inflammatory and pro-inflammatory cytokines and transforming growth factor-β (TGF-β).