Introduction: Night shift is one of the most frequent reasons for disturbance of circadian rhythm, altering biological functions and can negatively affect physical, psychological well-being and work performance. Aim of work: To evaluate the associations between circadian rhythm differences in shift-working nurses by using serum cortisol level and depression. Materials and Methods: a cross-sectional study was conducted by means of anonymous self-administered questionnaire for one hundred ninety-two nurses working at Menoufiya University Hospital, Shebin Al-Kom City, Egypt. The study involved 96 nurses working night-shifts and 96 in day-shifts. To assess their depression levels, self-rated depression scale (SDS) was used. Serum cortisol level was measured at 8:00 am for all respondents. Results: The response rate was 97%. A significant increase in serum cortisol level was observed among night-shift respondents compared to day-shift ones. Among night-shift workers, older nurses with more years of employment and higher cortisol levels were significantly more prone to develop depression (P<0.001). Conclusion: Nurses working night-shifts need special attention due to the higher risk of depression and possibly other undesirable health effects. Systems for early identification of nurses with depression and for providing environments allowing nurses at work to take rest and relax in the early morning after night shift are necessary.