Nightshift work involving circadian disruption has been associated with breast cancer risk. A role in prostate cancer is also suspected but evidence is limited. We investigated the association between nightshift work and prostate cancer incidence in the Prostate Cancer and Environment Study (PROtEuS), a population-based case-control study conducted in 2005-2012 in Montreal, Canada. Participants were 1,904 prostate cancer cases (432 high-grade cancers) and 1,965 population controls. Detailed work schedules for each job held at least 2 years (n =15,724) were elicited in face-to-face interviews. Nightshift work was defined as having ever worked ≥3 hours between midnight and 5:00 a.m., for ≥1 year, ≥3 nights/month. Unconditional logistic regression estimated odds ratios and 95% confidence intervals for the association between nightshift work and prostate cancer, adjusting for age, ancestry and education. No association was found between overall prostate cancer and nightshift work metrics including ever exposure, duration, intensity, cumulative exposure, rotating and early morning shifts. For none of the exposure indices was there evidence of heterogeneity in ORs between low- and high-grade cancers. Sensitivity analyses restricting exposures to ≥7 nights/month or considering the screening history yielded similar results. Our findings lend no support for a major role of nightshift work in prostate cancer development.