Background: To identify stress associated factors for vitamin D deficiency (VDD) in healthy Jordanian people based on serum 25(OH)D levels. Design: Prospective cohort study. Methods: Three hundred and seventy-one Jordanian men and women aged 17-52 years, who were identified as VD deficient 25(OH)D <30 ng/mL, were eligible to participate in the study. Serum vitamin 25(OH) D was measured using chemiluminescent immunoassay. Cortisol, parathyroid hormone, calcium, phosphate, fasting lipid profile, and blood glucose were also analyzed. Questionnaires were used to collect lifestyles parameters. Anthropometric parameters including: body mass index (BMI), waist (W) and hip (H) circumferences, W/H ratio (WHR) were also calculated. Results: The vast majority (91%) of the participants had vitamin D deficiency (25-(OH) D <30 ng/mL). Positive correlations were observed between vitamin D deficiency and the following anthropometric parameters in all study sample; gender (P=0.010), height (P=0.22), height/hip ratio (P=0.015) and waist/hip ratio (P=0.013). Lifestyle parameters that indicated very weak positive correlations with VDD were number of family members (P=0.011) and insufficient exposure to sunlight (P=0.023). The following clinical parameters showed weak or very weak correlations with VDD; serum cortisol (r=0.318), low density lipoprotein (r=0.246) and total cholesterol (r=0.133). Skin color and water pipe tobacco smoking were added to the multivariable stepwise regression analyses as they have been weakly correlated with VDD. These predictors together explained only 12.2% of the variance in serum cortisol levels in the VDD study sample. Conclusion: A weak positive association between VDD and elevated serum cortisol was observed in this study. Subcutaneous changes may be involved in that association but further studies are needed to clarify a potential role for adrenocorticotropic hormone (ACTH).