Objective: To investigate the regulatory effect of butyrate on intestinal epithelial barrier function during enterovirus A71 (EV71) infection and to explore the underlying molecular mechanisms. Methods: An in vitro intestinal epithelial barrier model was established using a Caco-2/HT29-MTX co-culture in Transwell inserts, and an EV71 infection system was subsequently developed. The cells were divided into a control group, an EV71 group, an EV71 + butyrate group, an ANT (GPR41/43 antagonist) + EV71 group, and an ANT + EV71 + butyrate group. Western blot was used to detect the protein expression of GPR41, GPR43, and the tight junction proteins Occludin, Claudin-1, and ZO-1. The secretion level of mucin 2 (MUC2) was measured by ELISA. RT-qPCR was used to measure EV71 RNA levels in the lower chamber of the Transwell system. Results: Compared with the control group, the EV71 group showed decreased expression levels of GPR41, GPR43, and the tight junction proteins Occludin, Claudin-1, and ZO-1 (P<0.05), along with a marked reduction in MUC2 secretion (P<0.05). Compared with the EV71 group, the EV71 + butyrate group exhibited increased expression of the above proteins and elevated MUC2 secretion levels (P<0.05). Compared with the EV71 + butyrate group, the ANT + EV71 + butyrate group exhibited lower expression of GPR41, GPR43, Occludin, Claudin-1, and ZO-1, together with reduced MUC2 secretion (P<0.05). RT-qPCR results showed that EV71 RNA expression was lower in the EV71 + butyrate group than in the EV71 group (P<0.05), whereas it was higher in the ANT + EV71 + butyrate group than in the EV71 + butyrate group (P<0.05). Conclusion: Butyrate may enhance intestinal epithelial barrier function and reduce EV71 transepithelial passage through GPR41/43-related signaling, possibly by upregulating tight junction protein expression and promoting MUC2 secretion.