This study investigated the effect of vitamin D supplementation in patients with dry eye syndrome (DES) refractory to conventional treatment with vitamin D deficiency. A total of 105 patients with DES refractory to conventional treatment and vitamin D deficiency that was treated with an intramuscular injection of cholecalciferol (200,000 IU). Serum 25-hydroxyvitamin D (25(OH)D) levels were measured. Eye discomfort was assessed using ocular surface disease index (OSDI) and visual analogue pain score (VAS). Tear break-up time (TBUT), fluorescein staining score (FSS), eyelid margin hyperemia, and tear secretion test were measured before treatment, and 2, 6, and 10 weeks after vitamin D supplementation. Mean serum 25(OH)D level was 10.52 ± 4.61 ng/mL. TBUT, and tear secretion test showed an improvement at 2 and 6 weeks after vitamin D supplementation compared to pretreatment values (p < 0.05 for all, paired t-test). Eyelid margin hyperemia and the severity of symptoms showed improvement at 2, 6, and 10 weeks after vitamin D supplementation (p < 0.05 for all). Compared to pre-treatment values, FSS, OSDI and VAS were decreased at 2 weeks (p < 0.05 for all). In conclusion, vitamin D supplementation is effective and useful in the treatment of patients with DES refractory to conventional treatment and with vitamin D deficiency.