INTRODUCTION: Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer patients. Despite the benefits of CRT, its use in non-responder patients can be associated with increased toxicities and surgical resection delay. The identification of CRT response biomarkers, such as microRNAs, could improve the management of these patients. We have studied the microRNA expression in pretreatment endoscopy biopsies from rectal cancer patients treated with CRT to identify potential microRNAs able to predict CRT response and clinical outcome of these patients. MATERIAL AND METHODS: RNA from pretreatment endoscopy biopsies from 96 rectal cancer patients treated with preoperative CRT were studied. Pathological response was graded according to the tumor regression grade (TRG) Dworak classification. In the screening phase, 377 miRNAs were studied in 12 patients with extreme responses (TRG0-1 vs TRG4). The potential role as predictive biomarkers for CRT response, disease-free survival (DFS) and overall survival (OS) of the miRNAs identified in the screening phase were validated in the whole cohort. RESULTS: In the screening phase, an 8-miRNAs CRT-response signature was identified: let-7b, let-7e, miR-21, miR-99b, miR-183, miR-328, miR-375 and miR-483-5p. In the validation phase, miR-21, miR-99b and miR-375 emerged as CRT response-related miRNAs while miR-328 and let-7e emerged as prognostic markers for DFS and OS. Interestingly, ROC curve analysis showed that the combination of miR-21, miR-99b and miR-375 had the best capacity to distinguish patients with maximum response (TRG4) from others. CONCLUSIONS: miR-21, miR-99b and miR-375 could add valuable information for individualizing treatment in locally advanced rectal cancer patients.