BACKGROUND: The aim of this study was to describe whether maintaining sufficient serum vitamin D levels in people with knee osteoarthritis and baseline vitamin D insufficiency has an association with change in knee structures and symptoms over 2 years. METHODS: Participants (n = 413, mean age 63.2 years) with symptomatic knee osteoarthritis and vitamin D insufficiency were enrolled in a clinical trial. In all, 340 participants (82.3%) completed the study, with 25-hydroxyvitamin D [25(OH)D] measurements at baseline and months 3 and 24. Participants were classified as consistently insufficient [serum 25(OH)D ≤50 nmol/L at months 3 and 24, n = 45], fluctuating [25(OH)D >50 nmol/L at either point, n = 68), and consistently sufficient [25(OH)D >50 nmol/L at months 3 and 24, n = 226] groups. Knee cartilage volume, cartilage defects, bone marrow lesions, and effusion-synovitis volume were assessed using MRI at baseline and month 24. Knee symptoms were assessed at baseline and months 3, 6, 12, and 24 using the Western Ontario and McMaster Universities Arthritis Index. RESULTS: The consistently sufficient group had significantly less loss of tibial cartilage volume (β 2.1%; 95% confidence interval [CI], 0.3%, 3.9%), less increase in effusion-synovitis volume (β -2.5 mL; 95 CI%, -4.7, -0.2 mL), and less loss of Western Ontario and McMaster Universities Arthritis Index physical function (β -94.2; 95% CI, -183.8, -4.5) compared with the consistently insufficient group in multivariable analyses. In contrast, there were no significant differences in these outcomes between the fluctuating and consistently insufficient groups. Changes in cartilage defects, bone marrow lesions, and knee pain were similar between groups. CONCLUSION: This post hoc analysis suggests beneficial effects of maintaining vitamin D sufficiency on cartilage loss, effusion-synovitis, and physical function in people with knee osteoarthritis.