The extent of the association between vitamin D deficiency and knee osteoarthritis remains inadequately understood. This study aimed to elucidate the relationship between vitamin D levels and knee osteoarthritis through a cross-sectional analysis. This retrospective study involved an analysis of knee radiographs and serum 25-hydroxyvitamin D3 (25-(OH) vitamin D3) levels in a cohort of 3424 individuals (2901 women and 523 men). Knee osteoarthritis severity was evaluated using the Kellgren-Lawrence radiological scoring system. Of the participants, 49.2% (n= 1,683) were diagnosed with knee osteoarthritis. Among these patients, the levels of adjusted 25-(OH) vitamin D3 were significantly lower (p< 0.001). Regression analysis revealed a significant association between vitamin D deficiency and knee osteoarthritis, with an adjusted odds ratio (OR) of 1.7 (95% CI: 1.5-2.0; p< 0.001). Notably, a stronger association was observed between vitamin D deficiency and knee osteoarthritis in women under 65 compared to those aged 65 and above. The findings of this study indicate a higher prevalence of vitamin D deficiency in patients with knee osteoarthritis. Maintaining adequate serum 25-(OH) vitamin D3 levels may prevent knee osteoarthritis, especially in women below 65.