Abstract: Objective To explore the association of cytoplasmic p53 with autophagy and apoptosis of primary aortic endothelial cells (MAECs) exposed to heat stress. Methods Cultured mouse MAECs were exposed to heat stress induced by incubation at 43 ℃ for 2 h, with the cells in routine culture condition (37 ℃, 5% CO2) as the control group. All the cells were further incubated for 1, 3, 6 or 9 h at 37 ℃ before treatment with the autophagy inhibitor 3-MA (5 mmol/L), the autophagy inducer rapamycin (20 μmol/L), or the p53 inhibitor PFT (10 μmol/L) for 1 h. After the treatments, the cell viability was measured with CCK8 method, cell apoptosis analyzed by flow cytometry, and the mitochondrial membrane potential detected with flow cytometry with JC-1 staining; the subcellular localization of p53 and the autophagy- associated protein LC3-II was detected with immunofluorescence staining, and their protein expressions were analyzed using Western blotting. Results Compared with the control cells, MAECs exposed to heat stress showed significantly decreased viability (P<0.05). At 6 h after the exposure, the cells exhibited significantly decreased mitochondrial membrane potential with increased apoptotic rate (P<0.05). The cytoplasmic fraction of p53 expression decreased and its mitochondrial fraction increased gradually with time within 6 h after heat stress. Treatment with 3-MA further decreased the mitochondrial membrane potential and significantly increased the apoptotic rate of the exposed cells (P<0.05), while rapamycin obviously reversed these heat stress-induced cell injuries (P<0.05). PFT significantly enhanced the expression of LC3-II and also inhibited heat stress-induced mitochondrial membrane potential reduction and cell apoptosis (P<0.05). Conclusion Heat stress induces mitochondrial damage and apoptosis in MAECs possibly in relation with mitochondrial translocation of cytoplasmic p53 to result in autophagy inhibition.