Objective To analyze the changes of intestinal microflora and drug-resistant genes in jaundiced neonates receiving phototherapy, and to explore its clinical significance. Methods Metagenomic sequencing was used to analyze the changes of intestinal flora and drug-resistant genes in neonates with jaundice after phototherapy. Results There was no significant difference in the α- and β-diversity of the intestinal microflora abundance after 24 and 48 hours of phototherapy (P>0.05). At the species level, the relative abundance of some species increased, including Clostridium bolteae, Enterococcus faecium and Streptococcus thermophilus (P<0.01). The intestinal flora of jaundiced neonates carried a large number of drug-resistant genes, a total of 242 genes, mainly tetracycline, β-lactams, macrolides, sulfonamides and aminoglycosides, and more resistant genes of Escherichia coli were also found. With the prolongation of phototherapy time, some drug resistance genes changed significantly. The main increased resistance genes were β-lactam resistance gene (CTX-M-14), aminoglycoside resistance gene (aadA2), tetracycline resistance gene [tet(59)], efflux pump gene (efmA), quinolone resistance gene (QnrS2), carbapenemase gene (OXA-347) and sulfonamide resistance gene (dfrA1), while the abundance of mgrA in Staphylococcus aureus decreased after phototherapy, and the differences were significant (P<0.05). After 48 hours of phototherapy, the species abundance of the following intestinal microflora showed a significant positive correlation with drug resistance genes: Clostridium and aminoglycoside resistance gene [APH(3')-Ia], Bacteroides uniformis and β-lactam resistance gene (CfxA6), Enterobacter cloacae and fosfomycin resistance gene (UhpT), Streptococcus parahaemolyticus and macrolide resistance gene (mefA) (P<0.01); while the species abundance of Enterococcus faecalis was negatively correlated with aminoglycoside resistance gene [ANT(2") -Ia] (P<0.01). Conclusions Phototherapy can change the relative abundance of some bacterial species in the intestinal microflora of neonates and significantly increase the abundance of some drug-resistant genes. Whether the disruption of bacterial homeostasis is related to the adverse reactions of phototherapy and its clinical significance needs further study.