Background. Botulinum toxin type A (BTXA) has been reported to increase survival of critically ischemic skin flaps; however, the effect of BTXA in human dermal microvascular endothelial cells (HDMECs) remains to be investigated. This study aimed to investigate the protective effect of BTXA in HDMECs exposed to an in vitro model of ischemia/reperfusion injury. Methods. HDMECs were isolated from human upper eyelid tissue and were randomly divided into 3 groups: 1. control group: culture under normoxic conditions (95% air and 5% CO2); 2. hypoxia/reoxygenation (H/R) group: culture in a hypoxic incubator (94% N-2 + 5% CO2 + 5% 02) for 8 hours, followed by culture in saturated aerobic culture medium for 24 hours; and 3. BTXA group: treatment with BTXA for 12 hours before exposure to hypoxic conditions. Flow cytometry was used to analyze the apoptosis of HDMECs, and western blotting was used to detect the expression of apoptosis-related proteins. Results. H/R leads to severe injury in HDMECs, as evidenced by an increase in the percentage of apoptosis and an increase in expression of apoptosis-related proteins (Bax, cleaved-caspase-3, and cytochrome C). Moreover, H/R results in a decrease in expression of anti-apoptotic protein (Bcl-2), which can be significantly attenuated with BTXA treatment. Conclusion. BTXA protects against apoptosis in HDMECs exposed to an in vitro model of H/R-induced injury.