Ischemia-reperfusion (IR) injury is a major cause of clinical emergencies during and after surgical procedures. Propofol protects the heart from cardiovascular IR injury by inhibiting autophagy. MicroRNAs (miRNAs) participate in anesthetic-regulated cardiovascular injury. MiR-20b-5p targets unc-51-like autophagy activating kinase 1 (ULK1). Its role in propofol-modulated cardiovascular IR injury remains unclear, however. In this study, we used an in vitro model of hypoxia-reoxygenation (HR)-induced injury to human umbilical vein endothelial cells (HUVECs) to determine the protective effect of miR-20b-5p in cells preconditioned with propofol. We found that miR-20b-5p was significantly higher and ULK1 was lower in propofol-preconditioned HUVECs with HR injury than in HUVECs with HR injury only. Additionally, miR-20b-5p overexpression increased cell viability and repressed autophagy and apoptosis more in propofol-preconditioned HUVECs with HR injury than in HUVECs with HR injury only. A luciferase reporter assay confirmed the target reaction between miR-20b-5p and ULK1. Overexpression of ULK1 restrained the protective effect of miR-20b-5p in propofol-preconditioned HUVECs with HR injury. In conclusion, our results indicate that propofol inhibits autophagic cell death via the miR-20b-5p-ULKI axis and that ULK1 may be a therapeutic target for cardiovascular IR injury.