MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, which play important roles in animals by targeting mRNA transcripts for translational repression. Many recent studies have shown that miRNAs are involved in the control of muscle development. In this study, the expression levels of miR-221 in different tissues and during rabbit skeletal muscle satellite cells (SMSCs) differentiation were detected. Gene ontology term enrichment was used to predict the potential biological roles of miR-221. A synthetic miR-221 mimic and a miR-221 inhibitor were used to investigate the functions of miR-221 during SMSCs proliferation and differentiation to further verify the functions of miR-221 in muscle development. In this report, we compared the expression levels of miR-221 in different tissues. The expression levels of miR-221 were upregulated after the induction of differentiation, and then were gradually downregulated during SMSCs differentiation. Overexpression of miR-221 promoted SMSCs proliferation, whereas inhibiting expression restrained proliferation in the EdU and CCK-8 assays. In addition, overexpression of miR-221 led to a decline in the expression levels of the differentiation marker genes MyoG and MHC. miR-221 overexpression suppressed SMSCs myotube formation. On the contrary, inhibition of miR-221 promoted myotube formation. Our data showed that miR-221 increased SMSCs proliferation and decreased differentiation.