Background Direct breastfeeding hasmultiple health benefits. Some mothers need to express their breasts when directly breast feeding is unavailable after delivery . However, there is insufficient evidence regarding an effective method of breast milk expression in the absence of an infant's suction. We aimed to investigate the effects of different methods of breast milk expression on breastfeeding initiation, efficacy of breast milk extraction, and on the rate of exclusive breastfeeding at 6 months. Methods In this open-label, randomized controlled trial, the participants of mother-infant dyads separated immediately after birth for at least 6 hrs were recruited and randomly assigned (1:1:1) into three groups: (1) hand expression of breast milk; (2) sequential electrical breast pumping: and (3) simultaneous electrical breast pumping. The two primary outcome measures were the time required for initiation of breastfeeding and the milk volume expressed in a 10-minute session (direct breastfeeding in hand expression group) at 3 days and at 6 weeks; a secondary outcome was the proportion of exclusive breastfeeding at 4 and 6 months. Analysis was according to intention-to-treat. Results Between 2018 and 2019, we recruited 226 participants, of whom 154 were randomized. The time to breastfeeding initiation showed no difference between the three groups. At 3 days and at 6 weeks, the volume of breast milk produced by 10 minutes of sequential pumping or simultaneous pumping was less than that of hand pumping before direct breastfeeding, while the volume did not differ significantly between the two pumping groups. However, simultaneous breast pumping was more effective than sequential pumping both in volume and saving time in women who had established breastfeeding. The rate of exclusive breastfeeding at 4 months was 21.0%, and at 6 months 10.6%, and did not differ between the three methods of breast milk expression. Conclusions Hand expression, sequential breast pumping, or simultaneous breast pumping in absence of immediate infant suction after birth did not show differences on the time required to initiate breastfeeding, or on the rate of exclusive breastfeeding at 4 and 6 months. Trial registration This trial was registered with www.chictr.org.cn (ChiCTR1800018193) on 4 September 2018.