This experiment was conducted to investigate the effects and mechanism of selenium (Se) on antioxidant and immune function of bovine mammary epithelial cells (BMEC) damaged by nitric oxide (NO). The third-generation BMEC was randomly divided into eight treatments with six replicates. The BMEC in the control group was cultured in the medium without Se and diethylenetriamine/NO (DETA/NO) for 30 h. For the DETA/NO group and Se protection group BMEC were exposed to different concentrations of Se (0, 10, 20, 50, 100, 150, and 200 nmol/L) for 24 h, followed by treatment with DETA/NO (1000 μmol/L) for 6 h. Compared with the control group, DETA/NO decreased proliferation rate and activity of thioredoxin reductase (TrxR; P < 0.05). Additionally, DETA/NO decreased the gene expression of both nuclear factor-E2-related factor 2 (Nrf2) and TrxR, as well as the protein expression level of TrxR. However, the activity, and expression levels of inducible nitric oxide synthase (iNOS), as well as the concentration and gene expression level of interleukin-1β (IL-1β) and the concentration of NO significantly increased (P < 0.05). The gene expression levels of indexes related to the mitogen-activated protein kinase (MAPK) signaling pathway showed similar changes. Treatment of BMEC with Se significantly reversed DETA/NO-induced changes in a linear or quadratic dose-dependent manner (P < 0.05), with greatest benefit at 50 nmol/L. These data suggests that Se improves the antioxidant function of BMEC, and protects cells from DETA/NO-induced oxidative damage, primarily by enhancing the activity of TrxR and decreasing the concentration of NO through modulation of Nrf2 and MAPK signaling pathways.