Night shift work has been classified as a probable human carcinogen based on experimental studies and limited human evidence on breast cancer. Evidence on other common cancers, such as prostate cancer, is scarce. Chronotype is an individual characteristic that may relate to night work adaptation. We evaluated night shift work with relation to prostate cancer, taking into account chronotype and disease severity in a population based case-control study in Spain. We included 1095 prostate cancer cases and 1388 randomly selected population controls. We collected detailed information on shift schedules (permanent vs rotating, time schedules, duration, frequency), using lifetime occupational history. Socio-demographic and lifestyle factors were assessed by face-to-face interviews and chronotype through a validated questionnaire. We used unconditional logistic regression analysis adjusting for potential confounders. Subjects who had worked at least for one year in night shift work had a slightly higher prostate cancer risk (Odds Ratio (OR) 1.14; 95%CI 0.94, 1.37) compared to never night workers; this risk increased with longer duration of exposure (≥28 years: OR 1.37; 95%CI 1.05, 1.81) (p-trend=0.047). Risks were more pronounced for high risk tumors (D'Amico classification, Relative Risk Ratio (RRR) 1.40; 95%CI 1.05, 1.86), particularly among subjects with longer duration of exposure (≥28 years: RRR 1.63; 95%CI 1.08, 2.45) (p-trend=0.027). Overall risk was higher among subjects with an evening chronotype, but also increased in morning chronotypes after long-term night work. In this large population based study we found an association between night shift work and prostate cancer particularly for tumors with worse prognosis. This article is protected by copyright. All rights reserved.