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 specific-pathogen-free Sprague-Dawley rats were injected with bilirubin or normal saline, respectively, and human milk or formula milk was administered 24 hours later for intervention. The rats were divided randomly into four groups: 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 enzyme-linked immunosorbent assay.The intestinal microbiota were analyzed using 16S rDNA high-throughput sequencing. Results There was no significant difference in bilirubin values among the groups. Pseudomonas was negatively correlated with indirect bilirubin value(P<0. 05). The composition of the intestinal microbiota differed significantly between human milk and formula milk after gastric administration, with Firmicutes ( P<0. 01), Enterococci ( P<0. 05), being the main microbiota in the HN and HB groups, and Proteobacteria ( P<0. 001 ), Escherichia Shigella ( P<0. 01 ) and Acinetobacter(P<0. 01) being the main in the FN and FB groups. Conclusions Pseudomonas may be negatively associated with bilirubin, and the structure of the intestinal microbiota may differ in relation to human milk and formula feeding.