Objective: To investigate the effect of irradiation to anastomosis from preoperative radiotherapy for patients with rectal cancer by studying the pathological changes. Methods: In this retrospective study, patients enrolled in the FOWARC study from January 2011 to July 2014 in the Sixth Affiliated Hospital of Sun Yat-Sen University were included. In the FOWARC study, enrolled patients with local advanced rectal cancer were randomly assigned to receive either neoadjuvant chemo-radiotherapy or chemotherapy. Among these patients, 23 patients were selected as radiation proctitis (RP)group, who fulfilled these conditions: (1) received neoadjuvant chemo-radiotherapy followed by sphincter-preserving surgery; (2) developed radiation proctitis as confirmed by preoperative imaging diagnosis; (3) had intact clinical samples of surgical margins. Twenty-three patients who had received neoadjuvant chemo-radiotherapy but without development of radiation proctitis were selected as non-radiation proctitis (nRP) group. Meanwhile, 23 patients received neoadjuvant chemotherapy only were selected as neoadjuvant chemotherapy (CT) group. Both nRP and CT cases were selected by ensuring the basic characteristics such as sex, age, tumor site, lengths of proximal margin and distal margin all maximally matched to the RP group. Both proximal and distal margins were collected for further analysis for all selected cases. Microscopy slices were prepared for hematoxylin & eosin staining and Masson staining to show general pathological changes, and also for immunohistochemistry with anti-CD-34 as primary antibody to reveal the microvessel. Microvessel counting in submucosal layer and proportion of macrovessel with stenosis were used to evaluate the blood supply of the proximal and distal end of anastomosis. A modified semi-quantitative grading approach was used to evaluate the severity of radiation-induced injury. Either ANOVA analysis, Kruskal-Wallis rank-sum test or χ² test was used for comparison among three groups, and Mann-Whitney U test was used for comparison between two groups. Results: Compared to group of neoadjuvant chemotherapy only, patients receiving neoadjuvant chemo-radiotherapy had lower microvessel count in both proximal and distal margins (M(Q_R): proximal, 25.5 (19.6) vs. 50.0 (25.0), Z=3.915, P=0.000; distal, 20.5 (17.5) vs. 49.0 (28.0), Z=3.558, P=0.000), higher proportions of macrovessel with stenosis (proximal, 9.5% (23.8%) vs. 0, Z=3.993, P=0.000; distal, 11.5%(37.3%) vs. 0 (2.0%), Z=2.893, P=0.004), higher histopathologic score (proximal, 4.0 (2.0) vs. 1.0 (2.0), Z=6.123, P=0.000; distal, 5.0 (3.0) vs. 2.0 (1.0), Z=4.849, P=0.000). In patients receiving neoadjuvant chemo-radiotherapy, compared to nRP group, RP group had lower microvessel count in both proximal and distal margins (proximal, 19.0 (23.0) vs. 30.4 (38.0), Z=2.845, P=0.004; distal, 19.0 (13.0) vs. 30.0(29.1), Z=2.022, P=0.043), higher proportions of macrovessel with stenosis (proximal, 23.0% (40.0%) vs. 0(11.0%), Z=3.248, P=0.001; distal, 27.0% (45.0%) vs. 3.0% (19.0%), Z=2.164, P=0.030). Rate of anastomotic leakage for CT, nRP and RP group were 8.7% (2/23), 30.4% (7/23), and 52.2% (12/23), and the differences among three groups were statistically significant (χ²=10.268, P=0.007). Conclusion Radiation-induced injury existed on both margins of the resected rectal site after preoperative radiotherapy, and those diagnosed as radiation proctitis had more severe microvascular injury.