Neuronal PAS domain protein 4 (Npas4) is a brain-specific transcription factor whose expression is enriched in neurogenic regions of the brain. In addition, it was demonstrated that Npas4 expression is dynamic and highly regulated during neural differentiation of embryonic stem cells (ESCs). While these findings implicate a role for Npas4 in neurogenesis, the underlying mechanisms of regulation remain unknown. Given that growing evidence suggests that microRNAs (miRNAs) play important roles in both embryonic and adult neurogenesis, we reasoned that miRNAs are good candidates for regulating Npas4 expression during neural differentiation of ESCs. In this study, we utilized the small RNA sequencing method to profile miRNA expression during neural differentiation of mouse ESCs. Two differentially expressed miRNAs were identified to be able to significantly reduce reporter gene activity by targeting the Npas4 3’UTR, namely miR-744 and miR-224. More importantly, ectopic expression of these miRNAs during neural differentiation resulted in downregulation of endogenous Npas4 expression. Subsequent functional analysis revealed that overexpression of either miR-744 or miR-224 delayed early neural differentiation, reduced GABAergic neuron production and inhibited neurite outgrowth. Collectively, our findings indicate that Npas4 not only functions at the early stages of neural differentiation but may also, in part, contribute to neuronal subtype specification and neurite development.