Vascularized composite allotransplantation (VCA) is a viable reconstructive option for complex tissue defects. Although grafts with a large muscular component may be uniquely susceptible to ischemia-reperfusion (I/R) syndrome, the safe cold ischemia time in VCA has not been established. We investigated the effects of cold ischemia on innate immune response and recipient survival in a murine orthotopic hindlimb transplantation model. Surprisingly, mice receiving grafts exposed to 6 h or longer of cold storage demonstrated reduced survival and massive elevations in serum creatinine, blood urea nitrogen, and creatine kinase, compared with 1 h of cold storage recipients. This was accompanied by progressive increase in macrophage and neutrophil cell infiltration in muscle biopsy specimens, altered platelet endothelial cell adhesion molecule-1 expression, and ultimate renal injury. Multiplex immunoassay analysis identified 21 cytokines in serum and 18 cytokines in muscle biopsy specimens that are likely essential in the complex response to I/R-triggered injury in VCA. In conclusion, this study, in a mouse model of orthotopic hindlimb transplantation, is the first to document that prolonged cold ischemia triggers progressive I/R injury with vascular endothelial damage and may lead to irrecoverable local and remote organ damage. These experimental findings are important in guiding future therapies for human VCA recipients.