Abstract: To explore the miRNAs related to the myostatin（MSTN） gens involved in regulating the growth and development of skeletal muscle in animals, the leg and hip muscles of five MSTN knockout(MSTN^-/-) Luxi cattle（test group） and five wild-type Luxi cattle（control group） were collected in vivo. Total muscle RNA was extracted for miRNA sequencing, differentially expressed miRNAs in muscle samples between two groups of Luxi cattle were screened, base preference of miRNA sequence was analyzed, and target genes of significantly differentially expressed miRNAs were predicted, and then the target genes were analyzed for gene ontology（GO） function and Kyoto encyclopedia of genes and genomes（KEGG） signaling pathway enrichment. Finally, real-time fluorescence quantitative PCR was utilized to verify the accuracy of the miRNA sequencing data in the satellite cell model of bovine skeletal muscle with MSTN gene interference. The results showed that the preference of the first base of the miRNA sequence was different with the variation of length, and the preference of the base at different positions was different. A total of 761 differentially expressed miRNAs were identified in the test and control samples, including 12 significantly differentially expressed miRNAs（miR-2285l, miR-496, miR-12006, miR-6533, miR-450b, miR-6524, miR-410, miR-335, miR-2447, miR-147, miR-33a, miR-654） in the experimental group. Among them, there were 9 highly expressed miRNAs（miR-2285l, miR-12006, miR-6533, miR-450b, miR-410, miR-335, miR-147, miR-33a, miR-654） and 3 lowly expressed miRNAs（miR-496, miR-6524, miR-2447） in the test group compared to the control group.5 969 target genes and 9 280 target sites of significantly differentially expressed miRNAs were predicted. Target genes are mainly enriched in functions related to intracellular part, membrane-bounded organelle, protein binding, positive regulation of biological process and metabolic pathways such as kaposi’s sarcoma-associated herpesvirus infection, endocytosis and MAPK signaling pathway. The expression changes of all six miRNAs（miR-6533, miR-450b, miR-2285l, miR-654, miR-12006, miR-496） in the validation test were consistent with the trends of the sequencing results. The results indicated that the 12 significantly differentially expressed miRNAs might be involved in MSTN gene regulation of bovine skeletal muscle growth and development.