To analyze the association between prostate cancer (PCa) risk and night shift work, chronotype, and sleep duration in the context of a population-based case-control study of incident prostate cancer in Spain, a total of 465 PCa cases and 410 controls were analyzed. Selection criteria were: (i) age 40–80 years, and (ii) residence in the coverage area of the reference hospitals for ≥6 months before recruitment. Exposure variables were: (i) night shift work (permanent or rotating); (ii) chronotype: morning, neither, or evening (Munich ChronoType Questionnaire) and (iii) sleep duration according to the recommendations of the American National Sleep Foundation. PCa aggressiveness was determined according to the International Society of Urology Pathology classification. Adjusted odds ratios (aOR) and 95% confidence intervals (95% CI) were estimated using logistic regression models. Night shift work was associated with PCa, aOR = 1.47 (95% CI 1.02–2.11), especially for rotating night shifts, aOR = 1.73 (95% CI 1.09–2.75). The magnitude of the association between ever night work and PCa was higher in evening subjects with aOR = 3.14 (95% CI 0.91–10.76) than in morning chronotypes with an aOR = 1.25 (95% CI 0.78–2.00). Working night shifts, especially rotating night shifts, could increase PCa risk. This risk may be higher in people with an evening chronotype.