Increasing the content of alpha-linolenic acid in milk fat might be desirable to meet consumer concerns about dietary healthfulness. However, the rich content of polyunsaturated fatty acids (PUFAs) will influence the oxidative stability of milk fat. This experiment was carried out to determine the effects of infusion with different amounts of high-linolenic perilla fatty acid (HLPFA) emulsion into the duodenum of dairy cows on milk fatty acid profile and the susceptibility of milk fat to oxidation. In a crossover design, 4 multiparous Holstein cows were infused duodenally with increasing amounts (0, 40, 80, 120, or 160 g/day) of free fatty acids from HLPFA emulsion or with carrier alone. Continuous infusions (20 to 22 h/day) were for 7 days at each amount. Infusions were homogenates of HLPFA with 15 g/day of xanthan gum, 5 g/day sodium alginate, and 25 g/day Tween 80; controls received carrier only. The concentration of n-3 PUFAs, especially alpha-linolenic acid, in milk fat increased linearly as HLPFA infusion increased, but the saturated fatty acids decreased linearly. The milk production and the activity of superoxide dismutase, glutathione peroxidase, and catalase in milk tended to decrease quadratically. The milk fat percentage, however, tended to increase. The concentration of malondialdehyde increased quadratically in milk fat. Results suggest that infusion with HLPFA emulsion at varying amounts enhanced the content of n-3 PUFAs in milk fat over the length of experiment but decreased the oxidative stability of milk fat.