Neoadjuvant concurrent chemoradiotherapy (nCCRT) followed by total mesorectal excision has become the standard of care for advanced rectal cancer, but the most effective regimen of chemotherapeutic agents has not yet been determined. The purpose of this study is to determine the effect of Mitomycin-C (MMC) in nCCRT for rectal cancer. From 2000 to 2017, patients with rectal adenocarcinoma who received nCCRT followed by radical surgery were enrolled in our study. The patients were retrospectively separated into two groups according to nCCRT regimens (with or without MMC). Other factors related to cancer down-staging after nCCRT, disease-free survival (DFS) and overall survival (OS) were analyzed. One hundred ninety-five patients received radiotherapy (RT) + MMC + oral tegafur-uracil (UFUR), and 191 patients received RT + UFUR without MMC as neoadjuvant CCRT. Adding MMC might increase the down-staging rate (odds ratio [OR] = 1.520, p = 0.058), and down-staging had significant effect to improve OS (OR = 1.726, p = 0.002) and DFS (OR = 2.185, p < 0.001). The OS and DFS were improved in patients who received MMC, although this result did not reach a statistically significant difference. There was a higher incidence of low-grade toxicities in the MMC group, especially neutropenia, genitourinary side effects, and dermatological side effects ( p < 0.001). Adding MMC to the regimen of nCCRT for rectal adenocarcinoma is shown to increase tumor down-staging rate and improve disease-free and OS, although these benefits come at the cost of increased low-grade toxicities. Prospective randomized studies are needed to explore the role of MMC in nCCRT for rectal cancer.