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Glycogen synthase kinase 3β (GSK3β) has a vital role in the regulation of many cellular processes. However, the role of GSK3β in muscle cell differentiation in sheep remains unknown. In this study, we investigated the function of GSK3β in skeletal muscle satellite cells (SMSCs) of sheep. An overexpression of <i>GSK3β</i> significantly inhibited myotube formation as well as the mRNA levels of myogenic genes (<i>MyoD</i>, <i>MyoG</i>, <i>MyHC1</i>, and <i>MyHC2a</i>) in sheep SMSCs. SB216763 treatment had a time-course effect on the phosphorylation levels of sheep GSK3β. In addition, reducing the activity of GSK3β lead to the promotion of sheep SMSCs differentiation as well as the mRNA levels of myogenic genes (<i>MyoD</i>, <i>MyoG</i>, <i>MyHC1</i>, and <i>MyHC2a</i>). This study illustrated the function of GSK3β to inhibit myogenesis in sheep SMSCs, which provided evidence for studying the mechanisms involved in the regulation of sheep SMSCs differentiation by GSK3β.