Background Hormone replacement therapy (HRT) is commonly used and may affect the accuracy of mammographic screening. Methods We examined the sensitivity, specificity, and small-cancer detection rate according to HRT use in 103 770 women in Victoria, Australia, who attended first-round screening in 1994 and who did not have a personal history of breast cancer or a breast lump or a bloodstained or watery nipple discharge at the time of screening. BreastScreen Victoria provides mammography to women aged 40 years and older every 2 years. Unconditional logistic modelling was used to adjust for age, family history, and symptom status. Findings The sensitivity of screening mammography for a 2-year screening interval was lower in HRT users (64.8% [95% CI 58-72]) than nonusers (77.3% [74-81]). In the target group (50-69 years), the sensitivity was 64.3% (57-72) in HRT users and 79.8% (76-84) in non-users. Among women who were diagnosed with cancer during the 2-year screening interval, HRT users were more likely to have a false negative result than nonusers (odds ratio 1.60 [1.04-2.21]) after adjusting for potential confounding factors. Specificity was 0.6% lower in HRT users compared with nonusers. Among women who did not have cancer diagnosed in the interval, HRT users were more likely to have a false positive result (adjusted odds ratio 1.12 [1.05-1.19]). Interpretation We show that HRT use reduces the sensitivity of mammographic screening. In countries where HRT use is widespread, the reduction in sensitivity with HRT use may undermine the capacity of population-based mammographic-screening programmes to realise their potential mortality benefit.