Purpose: To investigate the effect of weekly single high dose vitamin D supplementation on serum 25-hydroxyvitamin D [25(OH)D], and non-specific musculoskeletal pain in female nurses. Methods: In this prospective study in Kashan/Iran, from April 1, 2014, through September 30, 2014, the 150 nurses with vitamin D deficiency received the weekly pearls of 50,000 units of vitamin D 3 for 10 weeks. The serum level of 25(OH)D was measured before and after supplement therapy. The subjects were also . Downloaded from www.worldscientific.com by 201.74.129.64 on 06/22/16. For personal use only.asked to complete the Extended Nordic Musculoskeletal Questionnaire. All analyses were conducted with SPSS version 16. Results: After 10 weeks of intervention there was 21:3 AE 9:8 ng/mL increase in 25(OH)D. The 82 nurses (54.7%) had 25(OH)D in normal range, while the 68 nurses (45.3%) were still vitamin D deficient. Weight could explain 15.4% increase in 25(OH)D. Before intervention 135 (90%), of nurses reported musculoskeletal pain in at least one region, after intervention this number decreased to 72.7%. There was a statistically significant improvement in musculoskeletal pain in neck, shoulders, upper back, lower back, hips/tights, knees, and ankles/feet after intervention. Conclusions: The weekly single high dose of vitamin D for 10 weeks could resolve vitamin D deficiency in about half of the patients. Patients with non-specific musculoskeletal pain might benefit from vitamin D supplementation.