Aim To investigate the effect of Crocin on hypoxia/reoxygenation injury of cortical neurons and its molecular mechanism. Methods The cortical neurons of SD rats were isolated and cultured, and the control group, model group, Crocin (50 mg/L) group, Crocin (50 mg/L)+lipopolysaccharide (TLR4 activator, 100 μg/L) group were set. The cortical neurons in control group were routinely cultured; the cortical neurons in model group were given hypoxia 4 h and reoxygenation 24 h; the cortical neurons in Crocin group and Crocin+lipopolysaccharide group were intervened for 24 h, and then the model was established. The neuronal viability and apoptosis rate were detected by CCK-8 method and Annexin V-FITC/PI double staining method. The levels of inflammatory factors in the culture medium were detected by ELISA method. The expression of toll-like receptor 4 (TLR4), nuclear factor-κB p65 (NF-κB p65), p-NF-κB p65, inhibitor α of NF-κB (IκBα), p-IκBα, high mobility group box protein B1 (HMGB1), cleaved Caspase-3,Bü lymphoma-2 gene (Bcl-2), Bcl-2-associated X protein (Bax) were detected by Western blot method. Results Compared with the control group, the activity of cortical neurons in the model group decreased significantly (P＜0.05); the apoptosis rate, the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 in the culture medium, the expressions of TLR4, HMGB1, cleaved Caspase-3 and the ratio of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, Bax/Bcl-2 increased significantly (P＜0.05). Compared with the model group, the activity of cortical neurons in Crocin group increased by 94.97% (P＜0.05); the apoptosis rate decreased by 65.80% (P＜0.05); the levels of TNF-α, IL-1β, IL-6 in the culture medium decreased by 61.86%, 78.34%, 63.42% (P＜0.05); the expressions of TLR4, HMGB1, cleaved Caspase-3 and the ratio of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, Bax/Bcl-2 decreased by 73.43%, 52.13%, 81.52%, 69.70%, 60.55%, 95.05% (P＜0.05). Lipopolysaccharide significantly reversed the regulatory effects of Crocin on hypoxia/reoxygenation injured cortical neurons (P＜0.05). Conclusion Crocin can inhibit the apoptosis and inflammatory response of hypoxia/reoxygenation injured neurons, and has protective effect on hypoxia/reoxygenation injury of cortical neurons, which mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway activation.