Results from previous prospective studies linking serum 25-hydroxyvitamin D (25OHD) with fracture risk have been inconsistent. The present study examined the relationship between serum 25OHD and risk of incident major osteoporotic fracture (hip, spine, radius, and humerus) in older U.S. adults. The study used a pooled cohort of 4749 men and women ages 65 years and older from the third National Health and Nutrition Examination Survey (NHANES III, 1988–1994) and NHANES 2000–2004. Incident fractures were identified using linked mortality and Medicare records that were obtained for participants from both surveys. Serum 25OHD values were measured by radioimmunoassay in both surveys. Cox proportional hazards models were used to estimate the relative risk (RR) of fracture by serum 25OHD level. There were 525 incident major osteoporotic fractures (287 hip fractures) in the sample. Serum 25OHD was a significant linear predictor of major osteoporotic fracture and significant quadratic predictor of hip fracture in the total sample and among those with less than 10 years of follow-up, but it was not related to risk of either fracture type among those with ≥10 years of follow-up. Major osteoporotic fracture risk was increased by 26% to 27% for each SD decrease in serum 25OHD among those with less than 10 years of follow-up. Serum 25OHD was significantly related to risk of major osteoporotic fractures as a group and to hip fracture alone in this cohort of older U.S. adults from NHANES III and NHANES 2000–2004. However, the predictive utility of serum 25OHD diminished after 10 years. In addition, the relationship appeared to be linear when major osteoporotic fracture risk was considered but quadratic when hip fracture risk was assessed. © 2013 American Society for Bone and Mineral Research.