Objective:To investigate the role and regulatory mechanism of miR-125b-1-3p in rotavirus replication.Methods:MA104 cells were infected with rotavirus after upregulation and down-regulation of miR-125b-1-3p, respectively. The expression of miR-125b-1-3p and the copy number of rotavirus were analyzed by RT-PCR. The effect of miR-125b-1-3p on the protein expression of rotavirus was analyzed by immunofluorescence. The expression of related proteins involved in the regulation of miR-125b-1-3p was analyzed by Western blot analysis.Results:After rotavirus infection, the expression level of miR-125b-1-3p was significantly up-regulated, the copy number of VP7 and NSP3 gene of rotavirus decreased after up-regulation of miR-125b-1-3p, and the copy number of VP7 and NSP3 gene of rotavirus was significantly increased after down-regulation of miR-125b-1-3p.The fluorescence number of rotavirus protein decreased after upregulation of miR-125b-1-3p expression level, and increased after down-regulation of miR-125b-1-3p expression level. The activity of PI3K/Akt pathway was inhibited 16 h after rotavirus infection, and the up-regulation of miR-125b-1-3p could inhibit the activation of PI3K/Akt pathway.Conclusions:MiR-125b-1-3p inhibits rotavirus replication by regulating the PI3K/Akt pathway. These results provide an experimental basis for exploring the specific regulatory mechanism between miR-125b-1-3p and PI3K/Akt pathway, and provide a target for anti-infection therapy of rotavirus.