Context The skeletal effects of vitamin D remain controversial and it is uncertain whether variation in serum 25-hydroxyvitamin D (25OHD) levels over time influences bone mineral density (BMD). Objective We evaluated longitudinal stability of serum 25OHD and associations with changes in BMD in participants aged 46-70 years at baseline. Methods We studied 3698 Busselton Healthy Ageing Study participants (2040 female) with serum 25OHD and dual-energy x-ray absorptiometry (DXA) BMD assessments at baseline and at similar to 6 years follow-up. Restricted cubic splines were used to evaluate associations between changes in 25OHD and BMD. Results Mean season-corrected serum 25OHD was 81.3 +/- 22.7 and 78.8 +/- 23.1 nmol/L at baseline and 6 years, respectively, and showed moderate correlation (intraclass correlation coefficient: 0.724). Significant predictors of change in 25OHD concentration (Delta 25OHD) included baseline 25OHD, change in body mass index and vitamin D supplementation at follow-up. Greater decline in serum 25OHD over time was associated with significantly greater reduction in BMD at total hip and femoral neck, but the magnitude of the differences was small (estimated differences 0.004 g/cm(2) and 0.005-0.007 g/cm(2), respectively, for lowest quartile of Delta 25OHD compared with higher quartiles, adjusted for sex, baseline BMD, 25OHD, and demographics). No significant associations between Delta 25OHD and lumbar spine BMD were observed. Increase in 25OHD levels was not associated with change in BMD. Conclusions In this predominantly vitamin D-replete middle-aged cohort, serum 25OHD showed moderate longitudinal stability. Declining serum 25OHD over time was associated with greater reduction in BMD at the total hip and femoral neck.