The aim of this study was to evaluate the relationship between 25-hydroxyvitamin D (25(OH)D) levels and carpal tunnel syndrome (CTS). 25(OH)D levels were checked in 108 consecutive patients with CTS symptoms and 52 healthy controls. All patients underwent nerve conduction studies and completed Boston Carpal Tunnel Questionnaire (BQ) symptom severity and functional status scales to quantify symptom severity, pain status and functional status. There were 57 patients with electrophysiological confirmed CTS (EP+ group) and 51 electrophysiological negative symptomatic patients (EP- group). 25(OH) D deficiency (25(OH)D < 20 ng/ml) was found in 96.1 % of EP- group, in 94.7 % of EP+ group and in 73.8 % of control group. 25(0H) D level was found significantly lower both in EP+ and EP- groups compared to control group (p = 0.006, p < 0.001, respectively). Although mean vitamin D level in EP- group was lower than EP+ group, statistically difference was not significant between EP+ and EP- groups (p = 0.182). BQ symptom severity and functional status scores and BQ pain sum score were not significantly different between EP+ and EP- groups. We found no correlation with 25(OH) D level for BQ symptom severity, functional status and pain sum scores. 25(OH) D deficiency is a common problem in patients with CTS symptoms. As evidenced by the present study, assessment of serum 25(OH)D is recommended in CTS patients even with electrophysiological negative results.