This study aimed to explore mechanisms of the effects of hydroxysafflor yellow A (HSYA) on neural stem cells (NSCs) after heat stress (HS). Rat NSCs cells were cultured at 42 °C to impose heat stress. Cell counting kit-8 and Edu assay were used to analyze NSC proliferation. Annexin V/PI apoptosis kit was used to detect NSC apoptosis. Expression and phosphorylation of autophagy and apoptosis-associated proteins were determined by western blotting. We showed that HSYA significantly promoted proliferation and attenuated apoptosis of NSCs after heat stress. HSYA also increased Bcl-2 expression but decreased the expression of Bax and cleaved caspase-3 in NSCs induced by heat stress. In addition, HSYA decreased p38 and Hsp27-78 phosphorylation and MK-2 expression after heat stress, which was consistent with NSCs treated with SB203850 treatment or p38 knockdown. Furthermore, we demonstrated that heat stress increased LC3-II expression and mTOR phosphorylation, and decreased the expression of p62 in NSCs, while HSYA, SB203850 treatment or p38 knockdown reversed these alterations. In conclusion, HSYA significantly reversed the apoptosis and autophagy of NSCs induced by heat stress (P < 0.05), via downregulating MK2 expression and p38 and Hsp27-78 phosphorylation.