Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and re-warming-induced injury by interrogating a pro-apoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3 knockout (KO) mice, and 3) cold preservation and re-warming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during re-warming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved post-preservation mitochondrial viability. Cold preservation resulted in decreased post-isolation islet yield in both wild type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3 KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3 KO islets from cold-preserved pancreata showed reduced HMGB1 (High-mobility Group Box 1 protein) expression and decreased levels of 4-hydroxynonenal (4HNE) protein adducts, indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was up-regulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after re-warming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/re-warming-mediated islet injury, possibly through modulating HMGB1-and oxidative stress-mediated injury to islets.