Objective The factors affecting the establishment of the neonatal gut microbiota during puerperium were studied.Methods Metagenomic technology was used to study the formation of the gut microbial ecosystem of four neonates in confinement during the puerperium， using different feeding methods， birth methods and genders.Results The diversity of neonatal gut microbiota increased significantly from birth to 30 d， and the classes with significant differences in neonatal gut microbiota in the first month were Bacteroides and Actinomyces. The proportion of lactobacilli in the gut of mixed-fed neonates， who were fed breastmilk and infant formula at intervals， was significantly lower than that of neonates who were breastfed. Breastfed neonates had lower gut flora diversity compared to mixed-fed neonates.Mixed-fed neonates had higher gut flora diversity compared to breastfed neonates，the abundance of Bacteroides， Enterobacter and Clostridium was higher than that of breastfed newborns. The order of bacteria with significant differences in the intestinal flora of the breastfeeding and mixed-feeding groups was Bacillus. Additionally， the intestinal flora composition of female infants in the first month was more abundant and evenly distributed than that of male infants.Conclusion Feeding mode， delivery mode， and gender can affect the composition of intestinal microorganisms in newborns during puerperium in varying degrees.