OBJECTIVE: To explore the effect of vitamin D supplementation on insulin resistance in a group of Iranian patients with polycystic ovary syndrome and vitamin D deficiency. METHODS: This was a clinical trial conducted in a tertiary medical center in Tehran, the capital city of Iran, from May 2015 to September 2015. The participants included 41 women between 20 and 40 years of age with polycystic ovary syndrome based on the Rotterdam criteria and vitamin D deficiency. The fasting blood glucose and insulin levels, as well as serum 25-hydroxyvitamin D and homeostasis model assessment of insulin resistance (HOMA-IR) levels were measured at baseline and two months post treatment with a single dose of 300,000IU intramuscular vitamin D RESULTS: The mean age of participants was 26.6±4.1. The serum level of 25-hydroxyvitamin D increased (5.7±1.77 to 16.34±8.99 ng/mL, p<0.001). The mean fasting blood glucose reading significantly decreased from 109.56±14.59mg/dL in pre-treatment to 103.71±13.72mg/dL post treatment (p=0.003). There was a significant decrease in the mean fasting serum insulin level from 8.52±5.48 mcU/mL before treatment with vitamin D to 7.07±5.03 (p=0.019) µU/mL after the treatment. The mean HOMA-IR, as a sign of insulin resistance, significantly decreased from 2.37±1.76 to 1.87±1.49, indicating less insulin resistance. CONCLUSIONS: A single injection of vitamin D significantly decreased serum insulin levels and insulin resistance among patients with polycystic ovary syndrome.