Objectives: The prevalence of knee osteoarthritis (OA) is increasing with the aging population and is exacerbated by the growing numbers of obese older adults. Low levels of Vitamin D, measured by serum 25-hydroxyVitamin D (25(OH)D), in older adults and obese individuals are correlated with several negative health conditions, including chronic pain. This cross-sectional study sought to examine the interactive influence of 25(OH)D levels and obesity on knee OA pain and functional performance measures. Methods: The sample consisted of 256 (63% female) racially diverse (55% black/African Americans) middle-aged and older adults (mean age 56.8 y). Blood was collected for analysis of 25(OH)D by high-performance liquid chromatography. Participants provided self-report regarding knee OA pain and underwent a lower extremity functional performance test. Results: Results demonstrated that obesity was associated with lower levels of 25(OH)D. Participants with adequate 25(OH)D levels reported significantly less knee OA pain compared with participants with deficient or insufficient levels, regardless of obesity status. Furthermore, there was a significant interaction between obesity and 25(OH)D levels for lower extremity functional performance, such that obese individuals with adequate 25(OH)D levels demonstrated better performance than those obese participants with deficient or insufficient 25(OH)D levels. Discussion: The mechanisms by which adequate 25(OH)D levels are associated with pain severity and improved function have not been completely elucidated. It may be that the pleiotropic role of biologically active 25(OH)D influences pain and pain processing through peripheral and central mechanisms. Alternatively, higher levels of pain may lead to reduced outdoor activity, which may contribute to both obesity and decreased Vitamin D. Thus, investigating Vitamin D status in obese and nonobese individuals with knee OA warrants further study.