Objectives: To assess the association between night work and 25-hydroxy vitamin D (25OHD) levels, and to evaluate effect of potential risk factors on 25OHD levels. Material and Methods: A total of 140 adult Jordanian employees were recruited. Demographic, lifestyle and working patterns data were documented through a well-structured questionnaire. Vitamin D status was assessed by measuring circulating concentrations of 25OHD. Results: Mean 25OHD level was 23.8 ng/ml. No significant difference was found in 25OHD levels between the summer and winter (p = 0.46), or between males and females (p = 0.35). The female night workers had significantly lower serum 25OHD levels compared to the female day workers (p = 0.01). No significant difference in serum 25OHD levels was found between the night and day male workers (p = 0.25). The number of night shifts/month was negatively correlated with 25OHD levels in both the males and females (p = 0.01 and p = 0.007, respectively). Age was positively correlated with 25OHD levels in both the males and females (p = 0.02 and p = 0.001, respectively). Body mass index was negatively associated with 25OHD levels in the whole sample (p = 0.03), but not within each gender group (p = 0.21 for the males and p = 0.09 for the females). Smoking had no significant association with 25OHD levels (p = 0.99 for the males and p = 0.22 for the females). Conclusions: Our results suggest that women working night shifts are at higher risk of 25OHD deficiency, and, consequently, of other health problems linked to 25OHD deficiency. Int J Occup Med Environ Health 2016;29(5):859–870