MiR-124 functions as a tumor suppressor and plays an important role in tumorigenesis. A common polymorphism (rs531564, C>G) in the pri-miR-124 has been recently studied in connection with cancer risk. The aim of the present study was to investigate the association between pri-miR-124 rs531564 polymorphism and the risk and clinicopathological characteristics of colorectal cancer (CRC). Two case-control studies involving 900 CRC patients and 1110 cancer-free controls showed that pri-miR-124 rs531564 polymorphism was significantly associated with the decreased risk of CRC in Xuzhou population [GG vs. CC: OR = 0.25, 95%CI = 0.09-0.67, P = 0.003; (CG+GG) vs. CC: OR = 0.73, 95%CI = 0.56-0.94, P = 0.01; GG vs. (CC+CG): OR = 0.27, 95%CI = 0.10-0.70, P = 0.004; G vs. C: OR = 0.70, 95%CI = 0.56-0.89, P = 0.003], Bengbu population [GG vs. CC: OR = 0.20, 95%CI = 0.04-0.90, P = 0.02; GG vs. (CC+CG): OR = 0.21, 95%CI = 0.05-0.95, P = 0.03; G vs. C: OR = 0.72, 95%CI = 0.54-0.98, P = 0.03] and pooled population [GG vs. CC: OR = 0.26, 95%CI = 0.11-0.59, P<0.001; (CG+GG) vs. CC: OR = 0.76, 95%CI = 0.62-0.93, P = 0.008; GG vs. (CC+CG): OR = 0.27, 95%CI = 0.12-0.62, P < 0.001; G vs. C: OR = 0.71, 95%CI = 0.59-0.85, P<0.001]. Additionally, pri-miR-124 rs531564 polymorphism was significantly associated with the decreased risk of poor differentiation and lymph node metastasis of CRC. Our results suggest that pri-miR-124 rs531564 polymorphism may be a genetic modifier for developing CRC. However, further studies are needed to validate our findings.