Lactose intolerance (LI) is a significant contributor to diarrhea in infants and young children. Recent studies suggest a potential link between rotavirus (RV) infection and alterations in gut microbiota, which may play a role in the mechanism of LI. However, the precise underlying mechanism remains unclear and requires urgent clarification. In this study, we combined insights from immunology and gut microbiome research to propose that the activation of the IL-22/pSTAT3/RegIIIγ signaling pathway, triggered by gut microbiota, may be involved. To investigate this, we established a LI animal model using 7-day-old BALB/c mice, which were infected with RV via oral gavage. The syndromes of mice were carefully recorded and compared during the intervention experiment. Notably, we measured the the expression levels of immunocytokines and phosphorylated signaling proteins, including IL-22, phosphorylated STAT3, and RegIIIγ. Additionally, we assessed the relationships between gut microbiota and these key elements. Our results indicated that RV indeed causes LI, as severe diarrhea was observed in the mice during the first three days of RV infection, subsiding after seven days. ELISA results revealed an increase in IL-22 levels, phosphorylated STAT3, and RegIIIγ, suggesting that the mechanism of LI associated with RV is linked to the classical IL-22/pSTAT3/RegIIIγ signaling pathway. Furthermore, our analysis of the connections between gut microbes and this signaling pathway indicated that "Bacteroidetes" and Firmicutes were significantly positively correlated with IL-22 and lactase, respectively. This finding implies that alterations in gut microbiota may serve as a potential switch for the IL-22/pSTAT3/RegIIIγ signaling pathway, leading to increased lactase levels. In summary, our study demonstrated that the development of LI is associated with the activation of the IL-22/pSTAT3/RegIIIγ signaling pathway alongside changes in gut microbes during RV infection. This provides valuable insights into the mechanisms underlying LI and its clinical treatment through moduLation of the gut microbiome.