The purpose of this investigation was to elucidate the regulation of inducible nitric oxide synthase (iNOS) expression by vascular endothelial growth factor (VEGF) in a muscle flap ischemia model in rats. The gracilis muscle flap model was chosen. Sixty adult male Sprague Dawley rats were randomly divided into two groups (n = 30). After 4 hours ischemia, the experimental group received VEGF treatment, and the control group was given saline in the same fashion. At time intervals of 0, 2, 6, 12, and 18 hours (n = 6 for each time interval) after injection, tissue samples were biopsied for reverse transcriptase polymerase chain reaction, routine hematoxylin-eosin staining, and CD31 immunohistochemical staining. The result showed that iNOS expression is increased in the gracilis muscle flap ischemia model in rats compared with the control group within 6 hours after ischemia (p < 0.05). In the VEGF group, iNOS expression increased rapidly over the first 2 hours, but no statistically significant difference was observed at 12 and 18 hours between the two groups (p > 0.05). We concluded that the application of VEGF could maintain the structure of capillaries and upregulate iNOS expression during the first 6 hours after ischemia in the ischemic muscle flap of a rat model. These findings may provide the evidence to study the mechanism of VEGF in improving flap survival.