Objective:To investigate the influence of pre-pregnancy body mass index (BMI), gestational weight gain and early feeding behavior on delayed onset of lactogenesis stage Ⅱ(DOL Ⅱ).Methods:This was a prospective study involving puerperae from Women's Hospital of Nanjing Medical University from March 2020 to June 2020. Demographic data and delivery data were obtained using questionnaires and breastfeeding behavior and milk secretion were followed up every day after delivery. According to whether the lactation initiation was longer than 72 h or not, all subjects were divided into DOL Ⅱ group or non-DOL Ⅱ group. Differences in general condition and breastfeeding between the two groups were compared using independent sample t-test, Chi-square test, and Mann-Whitney U test. Multivariate logistic regression analysis was used to analyze the risk factors of DOL Ⅱ. Results:During the study period, a total of 390 cases were enrolled and 334 cases among them were analyzed due to lost of follow-up in 56 cases. The incidence of DOL Ⅱ was 19.2% (64/334). Univariate analysis showed that body mass index [lean:18.8% (12/64) vs 13.3% (36/270); fit:56.2% (36/64) vs 74.8% (202/270); overweight: 25.0% (16/64) vs 11.9% (32/270); χ2=9.78], mode of delivery [vaginal delivery: 37.5% (24/64) vs 52.6% (142/270); cesarean section: 62.5% (40/64) vs 47.4% (128/270); χ2=4.71], nipple type score in LATCH score [2.0 (1.0-2.0) vs 2.0 (2.0-2.0), U=－2.08], frequency of breastfeeding in 24 h [(6.3±3.0) vs (8.3±3.6) times per day, t=－3.94], adding formula within 24 h [71.9% (46/64) vs 56.3% (152/270), χ2=5.20] and the proportion of attending breastfeeding clinic during pregnancy [73.4% (47/64) vs 85.6% (231/270), χ2=5.44] were significantly different between the DOL Ⅱ and non-DOL Ⅱ groups (all P<0.05). Multivariate logistic regression analysis showed that pre-pregnancy overweight was an independent risk factor for DOL Ⅱ ( OR=2.240, 95% CI:1.020-4.918, P=0.044), either was pre-pregnancy overweight with appropriate gestational weight gain ( OR=5.595, 95% CI:1.492-20.985, P=0.011), while breastfeeding frequency within 24 h ( OR=0.867, 95% CI: 0.780-0.963, P=0.008) and attending breastfeeding clinic during pregnancy ( OR=0.377, 95% CI: 0.173-0.820, P=0.014) were independent protective factors for DOL Ⅱ. Conclusions:Women who were overweight before pregnancy are more likely to suffer from DOL Ⅱ. Extra guidance should be given to this population during early breastfeeding. Attending prenatal breastfeeding consultation and increasing the frequency of breastfeeding in the early postpartum period may prevent DOL.