Although high mammographic density is a risk factor for postmenopausal breast cancer, its etiology remains unclear. We examined whether serum and dietary cholesterol, which increase breast cancer risk and are involved in endogenous estrogen formation, were associated with increased mammographic density. We conducted a cross-sectional analysis of 302 healthy, sedentary postmenopausal women, aged 50-74 years, enrolled in the Alberta Physical Activity and Breast Cancer Prevention Trial between 2003 and 2006. In multiple linear regression models, no significant associations were observed between serum lipids and percent density or dense tissue area (Percent density: b (change in square root percent density per unit change in cholesterol level) = -0.06 (95%CI = -0.26 to 0.13); b = 0.06 (95%CI = -0.48 to 0.61); and b = -0.11 (95%CI = -0.33 to 0.10) for total cholesterol, high-, and low-density lipoprotein, respectively; similar results found for dense area). Alcohol consumption modified the association between triglycerides and percent density (>1 drink/day: b = -0.94 (95%CI = -1.79 to -0.10); = 1 drink/day: b = 0.19 (95%CI = -0.12 to 0.50); and no alcohol consumption: b = 0.15 (95%CI = -0.44 to 0.73). We found no evidence indicating any association between dietary and serum cholesterol levels and mammographic density.