Endothelial microvesicles (EMVs), released after endothelial cell (EC) apoptosis or activation, may carry many adverse signals and propagate injury by intercellular transmission. Caveolae are 50–100 nm cell surface plasma membrane invaginations involved in many pathophysiological processes. Recent evidence has indicated EMVs and caveolae may have functional effects in cells undergoing H/R injury. Propofol, a widely used anaesthetic, confers antioxidative stress capability in the same process. But the connection between EMVs, H/R, and caveolae remains largely unclear. Here, we found that H/R significantly increased the release of EMVs, the expression of CAV-1 (the structural protein responsible for maintaining the shape of caveolae), oxidative stress, and the mitochondrial damage, and all these changes were inhibited by propofol preconditioning. Interestingly, the caveolae inhibitor Mβ-CD strengthened the protective effect of propofol preconditioning. We further found that the release of EMVs is more significantly reduced under propofol preconditioning in the presence of the caveolae inhibitor Mβ-CD. EMVs released from H/R-treated cells caused a substantially increased mitochondrial and cellular damage to normal HUVECs after 4 hours of coculture. Thus, we conclude that inhibition of caveolae contributes to propofol preconditioning-suppressed microvesicles release and cell injury by H/R.