Although some evidence suggests a protective role of vitamin D against breast cancer, epidemiological findings are inconsistent. The current study investigated the relation of serum 25-hydroxyvitamin D (25(OH)D) levels with mammographic density. Baseline serum samples from 182 premenopausal women including 67 Caucasians and 74 Asians from a nutritional trial were analyzed for 25(OH)D. Mammographic density was assessed using a computer-assisted method. Serum 25(OH)D was not associated with mammographic density after adjustment for confounders (body mass index (BMI), age at mammogram, Asian ethnicity, age at first birth, parity and age at menarche). 25(OH)D levels were significantly lower in Asians than in Caucasians, but no significant ethnic differences in mammographic density were observed after adjusting for BMI. Although the current results indicate that serum 25(OH)D levels were not associated with mammographic density among premenopausal women, a possible protective effect of vitamin D against breast cancer may be mediated through other pathways.