Bacterial septicemia is a systemic inflammatory reaction mainly caused by the infection of Aeromonas hydrophila. Excessive development of inflammation may lead to septic shock or death in fish. A large number of studies have confirmed that miRNA is involved in the regulation of immune response after bacterial infection. To explore the regulatory mechanism of miR-462 in Ctenopharyngodon idella kidney (CIK) cells infected with A. hydrophila, the expression profiles of miR-462 upon A. hydrophila infection was detected by real-time quantitative PCR; the target genes of miR-462 were predicted by RNAhybrid software, and identified by dual-luciferase reporter assay system; in addition, the regulatory effect of miR-462 on downstream genes was analyzed. The results showed that the expression of miR-462 changed significantly after A. hydrophila infection, indicating that miR-462 participated in the regulation of immune response. Dual-luciferase reporter assay revealed that cx32.2, slc9a3.1 and tbk1 are the target genes of miR-462, which is further confirmed by the overexpression and inhibition experiments of miR-462. The expression of slc4a4a, tnfrs5, cxcl9 and cxcl11 were suppressed after miR-462 antagomir was transfected, which proved that miR-462 could affect the function of the downstream genes by targeting slc9a3.1 and tbk1. Our results may provide a theoretical basis for investigating the molecular mechanism of miR-462 regulating immune response in C. idella.