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Several recent studies investigated the role of the miR-29 family in muscle development. However, only a few studies focused on chicken skeletal muscle. In the present study, cell cycle, 5-ethynyl-2’-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and other assays indicated that miR-29b-1-5p can inhibit the proliferation of chicken primary myoblasts (CPMs); the western blot assay and immunofluorescence detection of myosin heavy chain (MyHC) demonstrated that miR-29b-1-5p can promote the differentiation of myoblasts. The functional enrichment analysis revealed that the target genes of miR-29b-1-5p may be involved in muscle tissue development, muscle organ development, and striated muscle tissue development, which are biological processes related to muscle development. The correlation analysis showed that these 6 genes, i.e., ankyrin repeat domain 9 (ANKRD9), lactate dehydrogenase A (LDHA), ankyrin repeat domain 9 (TCF12), ankyrin repeat domain 9 (FAT1), ankyrin repeat domain 9 (LIN9) and ankyrin repeat domain 9 (ITGB3BP), can be used as effective candidate target genes of miR-29b-1-5p. Moreover, miR-29b-1-5p inhibits the expression of ANKRD9by directly binding the 3′UTR of ANKRD9. Overall, these data indicate that miR-29b-1-5p inhibits the proliferation of primary chicken myoblasts, stimulates their differentiation, and is involved in the process of muscle development and that its effective target gene is ANKRD9. This study identified the molecular mechanism of miR-29b-1-5p in chicken muscle development.