Objective To investigate the effects of human milk on serum bilirubin levels and gut microbiota in neonatal rats with hyperbilirubinemia. Methods A total of 24 7-day-old SPF SD rats were randomly divided into 4 groups: bilirubin and normal saline were injected respectively, and 24 hours later, human milk or formula milk were administered for intervention, human milk-normal saline group (HN), human milk-bilirubin group (HB), formula milk-normal saline group (FN), and formula milk-bilirubin group (FB), samples were taken 72 hours later, and serum bilirubin values were detected by ELISA. 16S rDNA high-throughput sequencing was used to analyze intestinal microbiota. Results There were significant differences in the composition of intestinal microbiota between human milk and formula milk after gatric administration, and Firmicutes, Enterococci and Staphylococcus were the main microbiota in the HN group and HB group, while Proteobacteria, Escherichia coli-Shigella and Acinetobacter were the main ones in the FN group and FB group. There was no significant difference in bilirubin value between the groups, Pseudomonas was negatively correlated with indirect bilirubin value. Conclusion Changes in the structure of the intestinal microbiota in human milk and formula feeding, and Pseudomonas may be associated with bilirubin.