This study found that miR-27 is expressed in muscle and regulates muscle proliferation and differentiation. We explored the function and regulatory mechanism of miR-27b in goat muscle proliferation and differentiation. Compared with the Boer goat, higher expression of miR-27b was observed in all of the collected muscle tissues of Anhuai goat, excluding the kidney, whereas the opposite expression pattern was observed for Pax3, which showed lower expression in Anhuai goat. Expression of miR-27b decreased gradually during the proliferation of skeletal muscle satellite cells in Anhuai goat and increased during differentiation; however, the expression pattern of Pax3 was opposite. The regulatory activity of miR-27b demonstrated that miR-27b inhibited the proliferation of skeletal muscle satellite cells, but promoted their differentiation. Moreover, function research demonstrated that Pax3 negatively regulated myogenic differentiation of goat skeletal muscle satellite cells, but accelerated their proliferation. The results of a dual-luciferase reporter analysis showed that miR-27b directly targeted the 3'-untranslated regions of Pax3 mRNA, and western blot and immunofluorescence staining analyses showed that miR-27b inhibited expression of the Pax3 protein. In goats, miR-27b can regulate myogenic proliferation and differentiation by targeting Pax3.