Background Insufficient levels of serum 25-hydroxyvitamin D (25-OHD) influence the knee joint cartilage and lead to development and progression of knee osteoarthritis (OA). The purpose of this study was to determine the status of serum 25-OHD levels in patients with knee OA compared with controls. Methods A total of 148 patients with knee OA and 150 controls were studied. Serum 25-OHD was measured by the ELISA method and concentrations<20 ng/ml were considered as deficient levels. Mann-Whitney U test was used for comparisons of means and logistic regression analysis with calculation of odds ratio (OR) was applied to determine association. Results The mean ages of patients and controls were 60.2± 12.9 and 60.1±10.2 years, respectively (P=0.23). In the entire population the mean serum 25-OHD in OA patients was not significantly lower than controls (P=0.28), but in subgroup analysis the mean 25-OHD in OA patients aged <60 years was significantly lower than controls (23.8±18.8 vs. 34.5.±29.6 ng/ml, P=0.01). In this age group knee OA was significantly associated with serum 25-OHD deficiency which remained significant after adjusting for age and sex (adjusted OR=2.26, 95% CI 1.15-4.4, P=0.018). A greater association was observed in patients aged<55 years (OR=2.63, 95% CI 1.16-5.95, P= 0.01); whereas the association between OA and serum 25-OHD deficiency in patients aged ≥60 years did not reach a significant level. Conclusion These findings indicate a significant association between serum 25-OHD deficiency and knee OA in patients aged < 60 years and suggest serum 25-OHD measurement in any patient with symptoms suggestive of knee OA particularly at the initial stage of disease.