Background: Colorectal cancer (CRC) is caused by genetic aberrations. MACROD2 is commonly involved in somatic focal DNA copy number losses, in more than onethird of CRCs. In this study, we aimed to investigate the association of MACROD2 protein expression with clinical outcome in stage II and stage III colon cancer. Methods: Tissue microarrays (TMA) containing formalin-fixed paraffinembedded tissue cores from 386 clinically well-annotated primary stage II and III colon cancers were stained by immunohistochemistry and evaluated for MACROD2 protein expression. Disease-free survival (DFS) analysis was performed to estimate association with clinical outcome. Results: Loss of nuclear MACROD2 protein expression in epithelial neoplastic cells of stage III microsatellite stable (MSS) colon cancers was associated with poor DFS within the subgroup of 59 patients who received 5-fluorouracil (5-FU)-based adjuvant chemotherapy (p=0.005; HR=3.8, 95% CI 1.4-10.0). Conclusion: These data indicate that low nuclear expression of MACROD2 is associated with poor prognosis of patients with stage III MSS primary colon cancer who were treated with 5-FU-based adjuvant chemotherapy.