Ischemia-reperfusion injury is often the final and irreversible factor causing flap failure in microsurgery. The salvage of a microsurgical flap with an ischemia-reperfusion injury contributes to the success of microsurgical flap transfers. Activated protein C (APC)， a serine protease with anticoagulant and anti-inflammatory activities， has been shown to improve ischemic flap survival. To date， APC has yet to be applied to models of free flap with ischemia-reperfusion injury. In this study， we aimed to investigate the effect of APC on gracilis flap ischemia-reperfusion injury induced by gracilis vessels clamping and reopening. Sixty male Sprague-Dawley rats were randomly divided into 2 groups. After 4 hours of clamping for ischemia， flaps were reperfused and recombinant human APC (25 μg/kg) or saline was injected in the flaps through pedicles. At 0， 1， 4， 18， and 24 hours after injection (n = 6 for each time point)， the tissue samples were harvested. The muscle viability at 24 hours in saline group was 54.8% (15.1%)， whereas the APC-treated group was 90.0% (4.3%) (P < 0.05). The induced nitric oxide synthase (iNOS) mRNA expression increased with the time after reperfusion， which were 0.93 (0.25) to 2.09 (0.22) in saline group， and 0.197 (0.15) to 0.711 (0.15) in the APC-treated group. iNOS mRNA expression in the APC-treated group was significantly higher than the saline group at 1， 18， and 24 hours (P < 0.05). Numerous inflammatory cells were observed infiltrating and invading the muscle fibers in the saline group more than the APC-treated group. Increased number of polymorphonuclear cells was also noted in the saline group compared with the APC-treated group (P < 0.05). In conclusion， APC treatment can significantly attenuate ischemia-reperfusion injury and increase the survival of the free flap through down-regulating iNOS mRNA expression and reducing the inflammatory cells. Further research is still needed to be done on various mechanisms in which APC is protective to prevent tissue damage.