Prior reports have indicated that defective mismatch repair (MMR) has a favorable impact on outcome in colorectal cancer patients treated with surgery, immunotherapy, or adjuvant chemotherapy. However, the impact of MMR status on response to neoadjuvant radiotherapy in rectal cancer is not well understood. Here we report that dMMR was associated with improved disease-free survival (DFS) (  = 0.034) in patients receiving neoadjuvant chemotherapy (NCT). Patients with dMMR tumors who received neoadjuvant chemoradiotherapy (NCRT) achieved significantly worse DFS (  = 0.026) than those treated with NCT. Conversely, NCRT improved DFS (  = 0.043) in patients with pMMR tumors, especially for stage III disease with improved DFS (  = 0.02). The presence of dMMR was associated with better prognosis in rectal cancer patients treated with NCT. NCT benefited patients with dMMR tumors; while NCRT benefited patients with stage III disease and pMMR tumors. Patients stratified by MMR status may provide a more tailored approach to rectal cancer neoadjuvant therapy.