Purpose Radiotherapy-induced gut toxicity (RIGT) is associated with diarrhoea, pain and rectal bleeding and can occur as an acute or chronic toxicity. The microvasculature has been shown to be altered in the development of RIGT; however, the features are not yet characterized. We hypothesized that apoptosis of microvascular cells would occur early in the gastrointestinal tract following fractionated irradiation, followed by late microvascular changes, including sclerosis and telangiectasis. Methods Female Dark Agouti rats were treated with a 6-week fractionated radiation schedule of 3 × 2.5 Gy doses per week localized to the abdomen. At 3, 6 and 15 weeks, the intestines were assessed for markers of acute and chronic injury including morphological changes, collagen deposition, apoptosis and proliferation. Results Apoptosis of microvascular cells significantly increased at 6 and 15 weeks in the jejunum ( p  = 0.0026 and p  = 0.0062, respectively) and at 6 and 15 weeks in the colon ( p  < 0.0001 and p  = 0.0005, respectively) in rats receiving fractionated radiation to the abdomen. Histopathological changes of the colon microvasculature were also seen from week 3, including thickening of the lamina propria and dilated, thickened, telangiectatic vessels. Conclusions Findings of this study provide evidence of regional and timing-specific changes in the intestinal microvasculature in response to fractionated radiotherapy which may play a role in development of both acute and chronic RIGT.