This study aims to investigate the association between vitamin D levels and distal femoral cartilage thickness in healthy subjects. Eighty patients who were admitted to our outpatient clinic between May and July 2013 were classified into three subgroups according to their 25-OH vitamin D levels of < 10, 10-20, and a parts per thousand yen20 ng/mL. Distal femoral cartilage thickness was measured from the midpoints of the right medial condyle (RMC), right lateral condyle (RLC), right intercondylar area (RIA), left medial condyle (LMC), left lateral condyle (LLC), and left intercondylar area (LIA) by using musculoskeletal ultrasound (US). The group with severe vitamin D deficiency (< 10 ng/mL) had thinner femoral cartilage thickness at LMC (p = 0.005). Positive correlations were determined only between vitamin D levels and US measurements in the severe vitamin D deficiency group at RLC (r = 444, p = 0.020), LMC (r = 357, p = 0.067), and LLC (r = 568, p = 0.002). Low levels of vitamin D seem to affect the femoral cartilage thickness, adversely. Further studies are necessary to ascertain the clinical relevance of this change in cartilage thickness and whether vitamin D supplementation can reverse the cartilage thinning process or the allied clinical symptoms in the course of knee osteoarthritis.