Abstract: To investigate the changes of miRNA expression profile of Ctenopharyngodon idella kidney cell line (CIK) before and after overexpression of the Ctenopharyngodon idella antimicrobial peptide Hepcidin (CiHep), and its molecular mechanism, the constructed recombinant expression plasmid pmCherry-N1-Cihep was transfected into CIK cells, and its optimal overexpression time was screened by detecting the transcription level of the gene and observing the fluorescence intensity of fusion proteinm Cherry-CiHep. CIK cell samples were collected at the optimal overexpression time point, and further analyzed for differentially miRNA expression profiles using high-throughput sequencing and qPCR techniques. The results showed that the optimal overexpression time of CiHep gene in CIK cells was 72h after transfection. 1850 and 2013 known miRNAs were identified as well as 1266 and 1347 new miRNAs were found from the two constructed sRNA libraries, respectively. Compared with the control group, 460 DEmiRNAs were screened in the overexpression group, among which 392 were significantly up-regulated and 68 were significantly down-regulated. GO annotation and KEGG pathway analysis of 5150 DEmiRNA target genes revealed that 436 target genes were GO annotated and mainly involved in biological processes such as cellular processes, biological regulation and single cell biological processes. 157 target genes were enriched in 54 KEGG pathways. miRNA-mRNA-immune interaction network analysis revealed that 29 DEmiRNAs and 23 target genes potentially mediated CiHep involvement in 13 immune-related signaling pathways including C-type lectin receptor, PI3K-Akt, and NOD-like receptor. The qPCR validation results of 12 DEmiRNAs were consistent with high-throughput sequencing results. The characteristics of miRNA expression profiles of CIK cells before and after CiHep overexpression were resolved in the study, and clarified pma-miR-199b-5p, dre-miR-15a-5p, novel_miR_317 and novel_miR_536 played a potentially important role in the immune regulatory network, which laid the foundation for an in-depth investigation of the molecular mechanisms of miRNA immune regulation induced by antimicrobial peptides in fish.