Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p was related to the growth of skeletal muscle. Here, we further report that miR-146b-3p is essential for the proliferation, differentiation, and apoptosis of chicken myoblast. Elevated expression of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. Besides, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can inhibit the PI3K/AKT pathway. Our study also showed that both AKT1 and MDFIC can promote the proliferation and differentiation while inhibit the apoptosis of myoblast in chicken. Overall, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts in the proliferation, differentiation, and apoptosis of myoblast in chicken.