The objective of this study was to assess the preclinical safety of a bioprosthetic cardiac valve in a large-animal model. The experimental pericardial valve was treated with a dye-mediated photooxidation process (PhotoFix®) designed to enhance durability, and to reduce bioreactivity and mineralization potential. Ten experimental (Sulzer Carbomedics PhotoFix® pericardial valves, 25 mm) and three control valves (Carpentier-Edwards® 2700, 25 mm) were implanted in the mitral position of 13 juvenile sheep. The PhotoFix valves had minimal leaflet calcification (mean 0.20 ± 0.12 µg/mg) at 150 days, while the control valves had massive thrombus and extensive mineralization of leaflets. In-vivo testing in juvenile sheep showed the PhotoFix pericardial valve to have no device-related mortality, no significant tissue degeneration, and minimal mineralization. In contrast, control valves showed significant leaflet mineralization that led to device-related death.