Background Evidence from animal studies suggests that the gradual rise in follicle stimulating hormone (FSH) during reproductive senescence may contribute to the change in adiposity distribution characteristic of menopause. The potential independent role the interrelationships of FSH and estradiol may play in postmenopausal adiposity changes are not well studied. Methods In a sample of 667 postmenopausal women from the Women’s Health Initiative Buffalo OsteoPerio Ancillary Study, we studied the associations of serum FSH and estradiol levels with dual x-ray absorptiometry (DXA)-derived adiposity measures via cross-sectional and longitudinal analyses (5-year follow-up). Results In cross-sectional analyses, FSH levels were inversely associated with all measures of adiposity in models adjusted for age, years since menopause, smoking status, pack years, and hormone therapy (HT) use; these associations were not influenced by adjustment for serum estradiol. In longitudinal analyses, the subset of women who discontinued HT over follow-up (n=242) experienced the largest increase in FSH (+33.9 mIU/mL) and decrease in estradiol (-44.3 pg/mL) and gains in all adiposity measures in unadjusted analyses. In adjusted analyses, an increase in FSH was associated with a gain in percent total body fat, total body fat mass, and subcutaneous adipose tissue. Conclusions While cross-sectional findings suggest that FSH is inversely associated with adiposity, our longitudinal findings suggest that greater increases in FSH were associated with greater increases in percent total body fat, total body fat mass and subcutaneous adipose tissue. Future studies are needed to provide additional insight into FSH-adiposity mechanisms in larger samples.