Ischemia and reperfusion provokes barrier failure of the coronary microvasculature through activation of the endothelial contractile machinery and formation of intercellular gaps, leading to myocardial edema development that jeopardizes functional recovery of the heart. In vivo studies have shown that cannabinoid receptor (CBR) activation protects reperfused hearts against reperfusion injury in terms of infarct size reduction and functional recovery. The aim of the present study is to investigate whether CBR activation with R1-Methanandamide (R1M; 10mM) can protect coronary endothelium against reperfusion induced intercellular gap formation and to identify the underlying mechanism. Cultured rat coronary endothelial monolayers were subjected to conditions of simulated ischemia (ANoxia 40min; pH6.4; no glucose) and reperfusion (NORMoxia 40min; pH7.4; with glucose). Stimulation of CBR's during reperfusion significantly reduced reperfusion-induced intercellular gap formation (gaps[a.U.]:control:313±18; R1M:213±12*; n=12–16; *p<0.05 vs. control), CB1 receptor blockade with AM251 [250nM] during reperfusion completely abolished the R1M mediated protection (gaps[a.U.]: R1M+AM251: 294±24#; n=14; #p<0.05 vs. R1M) whereas blockade of CB2 receptors with AM630[300nM] or TRPV receptors with SB366791[10mM] did not impair the protective effect. Gap formation under continued normoxic perfusion was insignificant. Application of the Rac1 inhibitor NSC23766 [100mM] during reperfusion decreased the R1M mediated protection significantly (gaps[a.U.]:control:364±41; R1M:231±18*; R1M+NSC23766:314±25#; n=5–10; *p<0.05 vs. control; #p<0.05 vs. R1M). In conclusion, activation of endothelial cannabinoid receptors protects the coronary endothelium against reperfusion-induced gap formation. This protection is mediated through CB1 receptors and subsequent Rac1 activation.