Neural stem cells (NSCs) are a class of self‑renewing and undifferentiated progenitor cells that retain the ability to differentiate to neurons, astrocytes and oligodendrocytes. MicroRNAs (miRNAs) are small noncoding RNAs that serve crucial roles in regulating a number of cellular processes, including cell proliferation, differentiation and apoptosis. Our previous GeneChip data indicated that the expression of miR‑329‑3p was increased in neurons compared with NSCs. However, whether miRNA‑329‑3p participates in regulating NSC function remains to be elucidated. In the present study, it was identified that the expression of miR‑329‑3p was upregulated in NSCs during neuronal differentiation, whereas expression of transcription factor E2F1 (E2F1), a putative target gene of miR‑329‑3p, was downregulated. Using luciferase reporter assays, it was confirmed that miR‑329‑3p regulated E2F1 expression. As differentiation has been demonstrated to limit the proliferative capacity of NSCs, the effects of miR‑329‑3p and E2F1 modulation on NSC proliferation were examined. Forced overexpression of miR‑329‑3p or RNA‑mediated silencing of E2F1 inhibited NSC proliferation, and overexpression of miR‑329‑3p also inhibited E2F1 expression. Notably, ectopic expression of E2F1 reversed the inhibition of NSC proliferation induced by miR‑329‑3p overexpression. These results indicated that miR‑329‑3p may serve crucial roles in regulating the proliferation of NSCs, at least in part via inhibition of E2F1 expression. These data improve the understanding of the microRNA‑mRNA regulatory network that controls NSC proliferation.