The authors' objective was to discern whether lifestyle or health-related factors were confounders, effect modifiers, or irrelevant with regard to understanding observational associations of serum 25-hydroxyvitamin D (25(OH)D) with colorectal and breast cancer. The authors conducted nested case-control studies of colorectal cancer (310 cases, 310 controls) and breast cancer (1,080 cases, 1,080 controls) in the Women's Health Initiative Calcium and Vitamin D Clinical Trial (1994-2005). Case-control matching factors included age, latitude, race/ethnicity, and blood collection date. Serum 25(OH)D was assayed in baseline fasting blood. Conditional logistic regression was used to estimate odds ratios for each cancer by serum 25(OH)D concentration, comparing the relative effects of successively adding body mass index, physical activity, and other health and lifestyle characteristics particular to each cancer. In models with matching factors only, low (vs. high) serum 25(OH)D was associated with a colorectal cancer odds ratio of 2.72 (95% confidence interval (CI): 1.55, 4.77) and a breast cancer odds ratio of 1.33 (95% CI: 1.02, 1.72). In multivariate-adjusted models for colorectal cancer, the association strengthened (OR = 4.45, 95% CI: 1.96, 10.10). However, in multivariate-adjusted breast cancer models, associations were no longer significant (OR = 1.06, 95% CI: 0.78, 1.43). Adjusting for health and lifestyle characteristics has differential effects depending on the cancer site; when modeling such relations, investigators should take these factors into account. © The Author 2012. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved.