Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1microg ml(-1). This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.