Abstract： Objective To determine the human milk insulin(HMI) concentrations of healthy,gestational diabetes mellitus (GDM) and gestational impaired glucose tolerance (GIGT) lactating mothers and their correlative factors and to explore the effect of HMI on growth and development of neonates. Methods HMI of colostrum and mature milk collected from 148 healthy lactating mothers and 46 GDM/GIGT mothers were determined by radioimmnuoassay. The intergroup HMI levels were compared by Wilcoxon test. Results (1) In the GDM/GIGT group, colostrum HMI was 28.81 μU/ml(13. 84-43.14 μU/ml), significantly lower than that of mature milk which was 57.50 μU/ml(36. 70-82. 73 μU/ml) (Z=-4. 828,P=0. 000). HMI of mature milk in the healthy group was 35.88 μU/ml(25.91-46.85 μU/ml), lower than in the GDM/GIGT group (Z=-2.874,P=0.004). ( 2 ) The colostrum HMI of the mothers underwent cesarean section was 23.64 μU/ml (14.90-38.51 μU/ml) lower than in the vaginal delivery ones which was 38.89 μU/ml(23.14-65.54 μU/ml)(Z= -4. 510, P= 0. 000). (3) HMI of mature milk in mixed feeding cases was 42.58 μU/ml (26.60-73.06 μU/ml),significantly higher than that of breast feeding ones which was 36.32 μU/ml(26.00-46.16 μU/ml)(Z=-2. 377,P=0.019). (4) Both HMI in colostrum and mature milk were positively correlated with maternal BMI (P＜0.05). Conclusions The mode of delivery significantly affects the HMI in colostrum. GDM/GIGT and the feeding patterns have some effects on HMI levels in mature breast milk. The HMI level is positively correlated with maternal BMI.