To further clarify and develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans and develop modifiable biomarkers of risk for colorectal cancer, we conducted a pilot, randomized, double-blind, placebo-controlled, 2×2 factorial clinical trial to test the effects of calcium and vitamin D3, alone and in combination, on key DNA mismatch repair proteins in the normal colorectal mucosa. Ninetytwo men and women with at least one pathology-confirmed colorectal adenoma were treated with 2.0 g/d calcium or 800 IU/d vitamin D3, alone or in combination, versus placebo over 6 months. Colorectal crypt overall expression and distribution of MSH2 and MLH1 proteins in biopsies of normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, MSH2 expression along the full lengths of crypts increased by 61% (P = 0.11) and 30% (P = 0.36) in the vitamin D and calcium groups, respectively, relative to the placebo group. The estimated calcium and vitamin D treatment effects were more pronounced in the upper 40% of crypts (differentiation zone) in which MSH2 expression increased by 169% (P = 0.04) and 107% (P = 0.13) in the vitamin D and calcium groups, respectively. These findings suggest that higher calcium and vitamin D intakes may result in increased DNA MMR system activity in the normal colorectal mucosa of sporadic adenoma patients and that the strongest effects may be vitamin D related and in the differentiation zone of the colorectal crypt.