Chemoradiation prior to surgery in locally advanced rectal cancer is the current standard therapy. Unfortunately, this is not effective in all rectal cancer patients and associated with severe side effects, thus, prognostic marker molecules predicting the effectiveness of radiation would be of great interest. Aim of this study was to investigate pathophysiological mechanisms underlying radioresistance. Therefore, FFPE rectal tumor (n=50) and control tissue (n=39) of non-responders and responders to chemoradiation were analyzed using LC-MS-based proteomics. As a result, 1683 proteins were robustly identified. Comparing tumor with corresponding control samples revealed 199 significantly regulated proteins with FTL, PCOLCE and RCN3 being most striking in tumor tissue. While CAECAM 1, 5 and 6 as well as MCM protein complex components were significantly up-regulated in tumor tissue of non-responders compared to all types of responders, ER-stress and autophagic activity-related proteins, namely HYOU1, PDIA4 and RAB1B were only found significantly regulated when compared to complete responders, suggesting their importance in radioresistance mechanisms. Analyzing not only tumor but also surrounding control tissue allowed us to highlight the contribution of stroma to radioresistance. This study demonstrates the suitability of FFPE samples for proteomic analysis and presents potential molecular mechanisms mediating resistance to chemoradiation in rectal cancer.