To address concerns over limitations in the clinical use of glutaraldehyde (GA) fixation in bioprosthetic heart valves, we manufactured novel, branched poly(ethylene glycol) tetraacrylate (PEG-TA) crosslinked valve leaflets and evaluated cytotoxic, thrombogenic, hemolytic, and anticalcification effects, thermal stability, and mechanical properties, in comparison to decellularized valves (control) and GA crosslinked valves. Thermal denaturation temperatures were higher for PEG-TA valve leaflets compared to control and GA crosslinked valves (p < 0.001). Leaflet hydrolyzation rate was lower for the PEG-TA group than for GA and control groups (p < 0.05). Superior cytocompatibility was found for PEG-TA group leaflets (MTT, p < 0.01. apoptosis assay, p > 0.05). No thrombogenesis was found in platelet activation tests (p < 0.0001). Hemolysis assays showed that PEG-TA leaflets would not cause damage to blood cells (p > 0.05). Excellent anticalcification properties were confirmed by von Kossa staining, western blot, and atomic absorption spectroscopy (p < 0.0001) in a rat subcutaneous embedding model. Finally, the novel PEG-TA crosslinked material exhibits improved mechanical properties as compared to GA crosslinked materials (tensile strength, p < 0.001, Young's modulus, p < 0.001). On the basis of all results presented, it is clear that the performance characteristics of PEG-TA crosslinked valve leaflets make PEG-TA crosslinked leaflets a promising alternative for the next generation of bioprosthetic heart valve. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 322–336, 2014.