We investigated the association between plasma 25(OH)D and the subsequent colorectal cancer incidence risk by a nested case-control study in The Japan Public Health Center-based Prospective Study, covering 375 newly diagnosed cases of colorectal cancer from 38 373 study subjects during a 11.5-year follow-up after blood collection. Two controls were matched per case on sex, age, study area, date of blood draw, and fasting time. In a conditional logistic regression model with matched pairs adjusted for smoking, alcohol consumption, body mass index, physical exercise, vitamin supplement use, and family history of colorectal cancer, plasma 25(OH)D was not significantly associated with colorectal cancer in men or in women. However, the lowest category of plasma 25(OH)D was associated with an elevated risk of rectal cancer in both men (odds ratio (OR), 4.6; 95% confidence interval (CI), 1.0-20) and women (OR, 2.7, 95% CI, 0.94-7.6), compared with the combined category of the other quartiles. Our results suggest that a low level of plasma 25(OH)D may increase the risk of rectal cancer.