Introduction: Working with night shift system can have a substantial negative influence on the worker's physical, psychological, and mental health as well as their ability to accomplish their professional duties. Vitamin D insufficiency is a worldwide problem. It has a negative impact on the health of those who are vulnerable. It is hypothesized that night work may affect circulating vitamin D3 levels due to reduced exposure to direct sunlight. Aim of Work: to elucidate the relation between night shift work and 25-hydroxy vitamin D3 status among hospital nursing staff Materials and Methods: It is a cross-sectional comparative study on the nursing staff working in hospitals in Ismailia. The participants' number was150 individuals (who included night shift, day shift and rotating shift workers), a questionnaire was addressed including: personal data, occupational history, nightshift work characteristics, food description of vitamin D2 intake sources ,Index of exposure to sunlight. The level of 25-hydroxyvitamin D3 in the blood serum was measured. Results: The study showed that 44% of night shift and 38% of rotating shift nursing staff were deficient in 25-hydroxyvitamin D3, 38% of night shift and 46% of day shift nursing staff were insufficient, while only 34% of day shift were sufficient. The results revealed a significant difference in serum vitamin D3 levels between nursing staff groups (p < 0.005). The day shift staff median serum vitamin D3 level was 18 (15, 24) ng/ml, while the night shift staff median level was 13 (9, 17) ng/ml. The number of night shifts worked per month, per year, and taking a sleep during the night shift were all predictors of vitamin D3 deficiency. Conclusion: individuals who work the night and rotating shifts had lower levels of vitamin D3 than those who solely work the day shift. Recommendations: Health education about sun exposure guidelines and vitamin D food sources is recommended for night and rotating shift nursing staff and also indoor workers. Nursing staff should be encouraged to take breaks to go outdoors for sunlight exposure and to consume adequate amounts of vitamin D-rich foods; and vitamin D supplements to maintain optimal vitamin D levels.