OBJECTIVE This study aimed to examine vitamin D status as a determinant for development of type 2 diabetes and deterioration of glucose homeostasis. RESEARCH DESIGN AND METHODS A random sample of the general population of Copenhagen, Denmark, was taken as part of the Inter99 study. Included were 6,405 men and women aged 30-65 years at baseline (1999-2001), with 4,296 participating in the follow-up examination 5 years later (2004-2006). Vitamin D was determined at baseline as serum 25-hydroxyvitamin D [25(OH)D]. Diabetes was defined based on an oral glucose tolerance test and a glycosylated hemoglobin (HbA(1c)) test. Secondary outcomes included continuous markers of glucose homeostasis. RESULTS The risk of incident diabetes associated with a 10 nmol/L increase in 25(OH)D was odds ratio (OR) 0.91 (95% CI 0.84-0.97) in crude analyses. The association became statistically nonsignificant after adjustment for confounders, with an OR per 10 nmol/L of 0.94 (0.86-1.03). Low 25(OH)D status was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis after adjustment for confounders. Fasting and 2-h glucose and insulin as well as the degree of insulin resistance increased significantly more during follow-up among those with low 25(OH)D levels compared with those with higher levels. CONCLUSIONS Low 25(OH)D status was not significantly associated with incident diabetes after adjustment for confounders. However, it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis, indicating that low vitamin D status could be related to deterioration of glucose homeostasis.