This experiment was conducted to study the effects of dietary soy isoflavone supplementation on the immunological function in the mammary of Chinese Holstein cows during late lactation. Twelve cows with similar parity, body weight and milk production were randomly assigned to four groups with three cows in each group. Cows in the control group (group D) were fed the total mixed ration (TMR) and the cows in the other groups were fed the TMR supplemented with 10 (group A), 20 (group B) and 30 mg/kg (group C) soy isoflavone, respectively. To further evaluate the influence of soy isoflavone on the dairy cows during late lactation, mammary mast cells were isolated from the cows and cultured in the media containing different concentrations of soy isoflavone, which were 0 (group D1), 0.25 (group A1), 0.50 (group B1) and 0.75 mg/mL (group C1). The results showed as follows: 1) the contents of estrogen (E2), 3, 5, 3’-triiodothyronine (T3) and thyroxine (T4) in the serum and milk in the experimental groups were higher in varying degrees than those in the control group in the mid-anaphase after feeding the soy isoflavone. 2) The content of surface-type IgA (sIgA) in the milk from the experimental groups were significantly increased in prophase (P<0.05), and then decreased to the same level as the control group. The sIgA content in the mammary gland of the experimental groups was higher than that in the control group (P<0.05). 3) Tumor necrosis factor α (TNF-α) content in serum and milk in the experimental groups was decreased and significantly lower than that in the control group in the anaphase (P<0.05). TNF-α content in the mammary gland of the experimental groups was significantly lower than that in the control group (P<0.05). 4) In the cell co-culture experiment, 0.50 mg/mL soy isoflavone could inhibit the mRNA expression of TNF-α in mammary mast cell (P<0.05). The results indicate that dietary soy isoflavone supplementation can improve the immunological function of mammary gland of dairy cows, and the optimal supplemental level is 30 mg/kg.［Chinese Journal of Animal Nutrition，2011，23（1）：112-121］