Proliferation of neural stem cells (NSCs) is required for development and repair in the nervous system. NSC amplification in vitro is a necessary step towards using NSC transplantation therapy to treat neurodegenerative diseases. Folic acid (FA) has been shown to act through DNA methyltransferase to stimulate NSC proliferation. To elucidate the underlying mechanism, the effect of FA on the methylation profiles in neonatal rat NSCs was assessed by methylated DNA immunoprecipitation (MeDIP) and methylated DNA immunoprecipitation-DNA microarray (MeDIP-Chip). Differentially methylated regions (DMRs) were determined by quantitative differentially methylated regions analysis, and genes carrying at least three DMRs were selected for pathway analysis. Gene network analysis revealed links with steroid biosynthesis, fatty acid elongation and the PI3K/Akt/CREB, neuroactive ligand-receptor interaction, Jak-STAT and MAPK signaling pathways. Moreover, Akt3 acted as a hub in the network, in which 14 differentially methylated genes converged to the PI3K/Akt/CREB signaling pathway. These findings indicate that FA stimulates NSC proliferation by modifying DNA methylation levels in the PI3K/Akt/CREB pathway.