Objective To investigate the effects of rat senescent astrocytes on the proliferation of neural stem cells using an H2O2 -induced senescent astrocyte model. Methods Primary cortical astrocytes and telencephalic neural stem cells were isolated by trypsin digestion from newborn and fetal rats, respectively. Rat astrocytes were incubated for 4 h with H₂O₂ to establish the senescent model. The supernatants of senescent cells cultured for 3 and 7 days were collected as conditioned medium, then added at a ratio of 1 ∶3 or 1 ∶2 to observe its effect on neural stem cell counts and neurosphere counts (proliferation). Normal astrocyte conditioned medium was used as control medium. Results The 3 d normal conditioned medium had no effect on the short-term proliferation of neural stem cells, but inhibited their long-term proliferation. The 3 d senescent conditioned medium inhibited the proliferation of neural stem cells. The 7 d normal conditioned medium promoted the short-term proliferation of neural stem cells and inhibited their long-term proliferation. The 7 d senescent medium inhibited the proliferation of neural stem cells, and the inhibitory effect was greater than that of normal medium. Conclusions Rat senescent astrocytes induced by H₂O₂ inhibited the proliferation of neural stem cells, and the inhibition of long-term proliferation was greater than that of normal astrocytes.