Objective: This study was designed to investigate the effect of diet supplementation with rubber seed oil and flaxseed oil on serum fatty acids profile, oxidation stability of serum and milk, and immune function of dairy cows. Methods: Forty-eight mid-lactation Holstein dairy cows were randomly assigned to one of four treatments for 8 wk, including basal diet (CON) or the basal diet supplemented with 4% rubber seed oil (RO), 4% flaxseed oil (FO) or 2% rubber seed oil plus 2% flaxseed oil (RFO) on a dry matter basis. Results: Compared with CON, all the oil groups increased the levels of trans-11 C18:1 (vaccenic acid), cis-9, trans-11 C18:2 (conjugated linoleic acid, CLA) and C18:3 (α-linolenic acid, ALA) in serum. Both the activity of glutathione peroxidase and catalase in serum and milk in oil groups were decreased, which were negatively correlated with the levels of cis-9, trans-11 CLA and ALA. The concentrations of proinflammatory factors (tumor necrosis factor α and interferon γ) in serum of oil groups were lower than that from the CON cows. Conclusion: These results indicate that diet supplementation with RO or FO could alter serum fatty acid profile and enhance the immune function of dairy cows. However, the negative effect on milk oxidation stability should be considered when feeding these n-3 polyunsaturated fatty acid-enriched oils in dairy production.