Background: Night-shift work (NSW) has been suggested as a possible cause of breast cancer, and its association with mammographic density (MD), one of the strongest risk factors for breast cancer, has been scarcely addressed. This study examined NSW and MD in Spanish women. Methods: The study covered 2,752 women aged 45-68 years recruited in 2007-2008 in 7 population-based public breast cancer screening centers, which included 243 women who had performed NSW for at least one year. Occupational data and information on potential confounders were collected by personal interview. Two trained radiologist estimated the percentage ofMD assisted by a validated semiautomatic computer tool (DM-scan). Multivariable mixed linear regression models with random screening center-specific intercepts were fitted using log-transformed percentage ofMDas the dependent variable and adjusting by known confounding variables. Results: Having ever worked in NSW was not associated with MD =eb:0.96; 95% confidence interval (CI), 0.86-1.06]. However, the adjusted geometric mean of the percentage of MD in women with NSWfor more than 15 years was 25%higher than that of those without NSW history (MD>15 years:20.7% vs. MDnever:16.5%;eb:1.25; 95% CI,1.01-1.54). This association was mainly observed in postmenopausal participants (eb:1.28; 95% CI, 1.00-1.64). Among NSW-exposed women, those with 2 night-shifts per week had higher MD than those with 5 to 7 nightshifts per week (eb:1.42; 95% CI, 1.10-1.84). Conclusions: Performing NSW was associated with higherMD only in women with more than 15 years of cumulated exposure. These findings warrant replication in futures studies. Impact: Our findings suggest that MD could play a role in the pathway between long-term NSW and breast cancer.