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The aim of this research project was to test the effects of lifetime night work exposure on type 2 diabetes (T2DM) risk. The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) is a prospective cohort study of chronic conditions, such as diabetes and cardiovascular disease. The participants (N = 15105) were recruited (2008–2010) at five public universities and one research institute in six cities in Brazil. Participants from the first wave (2008–2010) were followed up for a mean of 3.8 years. Current analyses comprise 4671 women and 3965 men. Hazard ratios (HR) were estimated using Cox regression models. Crude T2DM incidence rates were 2.26 and 1.44 per 100 person-years, respectively, for women and men who reported ≥ 10 years’ working nights. In women, ≥ 10 years of night work was associated with a higher risk of T2DM (HR 1.46 [95% CI: 1.03; 2.08]), after adjusting for age, education, work hours, and BMI. The additional adjustment for physical activity attenuated the association (HR 1.36 [95% CI: 0.94; 1.96]). In men, the results were not statistically significant (HR = 0.65 [95% CI: 0.40; 1.07]). The findings indicate the effects of lifetime night work on T2DM incidence seem to be greater among women than men.