UNLABELLED: Rotavirus (RV), a major cause of childhood diarrhea, damages intestinal epithelium and may induce lactose intolerance in some patients. Increasing studies have emphasized the importance of dysregulated gut microbiome community in diseases. In this study, fecal samples from rotavirus-infected children (n = 48) and healthy controls (n = 29) were analyzed based on 16S rRNA gene sequencing and clinical data. The rotavirus-infected group was further stratified by lactose tolerance status to investigate the distinctive features of the gut microbial communities. Our study verified a shift in gut microbiome constitution and abundance between the feces of healthy donors and rotavirus infection children. Notably, Agathobacter, Streptococcus, Veillonella, and Roseburia were significantly increased in rotavirus infection patients, while the relative abundance of the Ruminococcus gnavus group was decreased. Analyses of alpha diversity unveiled significant differences in the abundance of gut microbiota between rotavirus infection patients with or without lactose intolerance. In particular, the abundance of Parasutterella, Lachnospiraceae NK4A136 group, Morganella, and Veillonellaceae unclassified decreased significantly in rotavirus infection patients with lactose intolerance. Further, PLR, an inflammatory hematological ratio, was significantly elevated in rotavirus infection patients with lactose intolerance. CONCLUSION: Overall, our study indicated the characteristic of change in diversity, taxonomy, and function of gut microbiota between rotavirus infection and healthy control in children, revealing the existence of dysbiosis in rotavirus infection. Further, our analysis identified the compositional uniqueness of gut microbiota between rotavirus infection patients with or without lactose intolerance, highlighting the diversity and taxonomy in gut microbiome and association with inflammatory state. WHAT IS KNOWN: • Rotavirus causing diarrhea in children worldwide, causing damage to the intestinal epithelium and lactose intolerance-related diarrhea in children. WHAT IS NEW: • Results demonstrated the characteristic changes of gut microbiome in rotavirus-infected children and further investigated the different gut microbiome composition between rotavirus infection-related lactose intolerance and lactose tolerance patients. • Further, the relationship between gut microbiome and inflammatory hematological ratios was explored.