Neurogenesis is a process of generating neural stem cells (NSCs) as functional neurons can be decreased after chemotherapy treatments. Methotrexate (MTX) is a folate antagonist that is used for cancer treatment but has negative effects, including oxidative stress, neuronal apoptosis and cognitive impairments. Hesperidin (Hsd), a flavonoid found in citrus fruits, has antioxidant and neuroprotection properties. This study investigated whether Hsd could attenuate impairments of hippocampal neural stem cells related to apoptosis induced by MTX. Spraque-Dawley rats (n = 24) were divided into 4 groups: (1) Vehicle group received propylene glycol (21 days) and 0.9% normal saline (day 8 and 15), (2) Hsd group received 100 mg/kg (21 days), (3) MTX group received 75 mg/kg (days 8 and 15) and (4) MTX+Hsd group received MTX, 75 mg/kg (day 8 and 15) and Hsd 100 mg/kg (21 days). Our results showed that MTX decreased hippocampal neural stem cells including SRY (sex determining region Y)-box 2 (SOX2) and nestin. MTX diminished vascular related (VR) Ki-67 positive cells in the hippocampus but not non-vascular related (NVR) Ki-67. Additionally, MTX reduced SOX2, nestin, postsynaptic density protein 95 (PSD-95) and B-cell lymphoma-2 family of proteins (Bcl-2), whereas Bax and caspase-3 were enhanced in the hippocampal tissues. Interestingly, co-treatment with Hsd and MTX revealed upregulation of SOX2, nestin and VR Ki-67 positive cells as well as elevated SOX2, nestin, PSD-95 and Bcl-2 proteins. Moreover, receiving both Hsd and MTX significantly suppressed increased Bax and caspase-3. These results confirm that Hsd can ameliorate MTX-induced impairments of hippocampal NSC proliferation and neuronal apoptosis.