Microcystin-LR (MC-LR) poses a serious threat to human health due to its hepatotoxicity. However, the specific molecular mechanism of miRNAs in MC-LR-induced liver injury has not been determined. The aim of the present study was to determine whether miRNAs are regulated in MC-LR-induced liver toxicity by using high-throughput sequencing. Our research demonstrated that 53 miRNAs and 319 miRNAs were significantly changed after 24 h of treatment with MC-LR (50 and 200 μg/kg, respectively) compared with the control group. GO enrichment analysis revealed that these target genes were related to cellular, metabolic, and single-organism processes. Furthermore, KEGG pathway analysis demonstrated that the target genes of differentially expressed miRNAs in fish liver were primarily involved in the insulin signaling pathway, PPAR signaling pathway, Wnt signaling pathway, and transcriptional misregulation in cancer. Moreover, we hypothesized that 4 miRNAs (miR-16, miR-181a-3p, miR-451, and miR-223) might also participate in MC-LR-induced toxicity in multiple organs of the fish and play regulatory roles according to the qPCR analysis results. Taken together, our results may help to elucidate the biological function of miRNAs in MC-LR-induced toxicity.