Vitamin D deficiency is one of the most preventable challenges worldwide. The aim of this study was to determine the prevalence of vitamin D deficiency among female nurses working at Children’s Medical Center Hospital in Tehran, Iran, due to the risk factor of being a notably long period indoors and the fact that their health status may have consequences on the process of patients’ treatment. A total of 114 female nurses who were at least 20 years old entered the study voluntarily, and a questionnaire was applied to collect information on lifestyle and other factors associated with vitamin D deficiency. A sample of blood was taken to measure 25-hydroxyvitamin D (25-OHD) and cut off value to indicate deficiency was considered below 10ng/ml, and the amounts of 10-29ng/ml were declared insufficient. The mean of 25-OHD was 11.7±9.3ng/ml. A total of 79 subjects (69.3%) had a deficient level of vitamin D, 28 subjects (24.6%) had an insufficient level and only 7 subjects (6.1%) had sufficient level of vitamin D. The deficiency was more noticeable in the age group of 26-35 years old. Prevalence of vitamin D deficiency had a significant correlation with younger subjects (P<0.001). There was no significant association among other factors such as body mass index (BMI), health status complications, regular exercise, and duration of sun exposure. High prevalence of vitamin D deficiency in the study population leads to emphasise the need to screen health care workers for vitamin D levels.