The aim of the present study was to determine if folate alters Notch signaling and cell proliferation in neural stem cells (NSCs). NSCs were isolated from neonatal rats and grown in serum-free suspension culture. The cells were identified as NSCs by their expression of immunoreactive nestin. Individual cultures were assigned to one of three treatment groups: vehicle control, low-dose folate group (Folate-L, liquid media contained 4 mg/L folate), or high-dose folate group (Folate-H, liquid media contained 40 mg/L folate). Proliferating cells were identified by labeling with 5-bromo-2'-deoxyuridine (BrdU). Cell proliferation was quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Gene expression of components of the Notch signaling system (Notch1, Hes1 and Mash1) was quantified by real-time polymerase chain reaction (PCR) assay. We observed that Nestin-positive NSCs grew as neurospheres in the serum-free suspension cultures. Folate increased the rate of cell proliferation compared to vehicle control (p<0.05). During cell proliferation, folate also increased Notch1 and Hes1 expression and decreased Mash1 expression compared to vehicle control (p<0.05). These results suggest that NSCs cultured from neonatal rats respond to folate with altered Notch signaling and increased cell proliferation.