Our previous study has shown that actin alpha 2 (ACTA2) is expressed in NSC and ACTA2 downregulation inhibits NSC migration by increasing RhoA expression and decreasing the expression of Rac1 to curb actin filament polymerization. Given that proliferation and differentiation are the two main characteristics of NSC, the role of ACTA2 downregulation in the proliferation and differentiation of NSC remains elusive. Here, the results demonstrated that ACTA2 downregulation using ACTA2 siRNA held the potential of inhibiting NSC proliferation using cell counting kit-8 (CCK8) and immunostaining. Then, our data illustrated that ACTA2 downregulation attenuated NSC differentiation into neurons, while directing NSC into astrocytes and oligodendrocytes using immunostaining and immunoblotting. Thereafter, the results revealed that the canonical Wnt/β-catenin pathway was involved in the effect of ACTA2 downregulation on the proliferation and differentiation of NSC through upregulating p-β-catenin and decreasing β-catenin due to inactivating GSK-3β, while this effect could be partially abolished with administration of CHIR99012, a GSK-3 inhibitor. Collectively, these results indicate that ACTA2 downregulation inhibits NSC proliferation and differentiation into neurons through inactivation of the canonical Wnt/β-catenin pathway. The aim of the present study is to elucidate the role of ACTA2 in proliferation and differentiation of NSC and to provide an intervention target for promoting NSC proliferation and properly directing NSC differentiation.