BACKGROUND: Female gender and increasing age are key risk factors for gallstone disease; therefore, postmenopausal women are at high risk. Estrogen increases cholesterol saturation of bile and may further increase gallstone risk, but population-based evidence is sparse. OBJECTIVE: Our objective was to examine the association between postmenopausal estrogen therapy and risk of gallstone disease and the impact of duration of treatment and use of opposing progestin. STUDY DESIGN: We conducted a population-based case-control study. Cases were postmenopausal women (defined as aged ≥45 years) with gallstone disease identified in the period 1996-2010. For each case, we selected ten population controls matched to cases by age and sex. We defined exposure as any use of estrogen (opposed and unopposed by progestin). Cases/controls were categorized as current estrogen users if their last prescription was redeemed <90 days before gallstone diagnosis (or corresponding date for controls); all other users were categorized as former users. The reference group consisted of cases/controls with no/rare estrogen use. SETTING: Medical databases covering the population of Northern Denmark (2.4 million inhabitants through the period 1996-2010). MAIN OUTCOME MEASURE: We used conditional logistic regression to compute adjusted odds ratios (ORs) and 95 % confidence intervals (CIs) of gallstone disease in women treated with estrogen. The ORs were adjusted for relevant comorbidity, other drugs known to influence gallstone risk, and parity. RESULTS: We identified 16,386 cases with gallstone disease and 163,860 controls. A total of 1,425 cases (8.7 %) and 8,930 controls (5.4 %) were current estrogen users, yielding an adjusted OR for gallstone disease of 1.74 (95 % CI 1.64-1.85) compared with non-users. The corresponding adjusted OR for former users was 1.35 (95 % CI 1.28-1.42). The results suggested a duration response for current users. Use of unopposed estrogen was associated with higher adjusted ORs than estrogen opposed by progestin. CONCLUSION: Postmenopausal estrogen therapy was associated with increased risk of gallstone disease in current and former estrogen users. Use of unopposed estrogen was associated with higher risk than use of estrogen opposed by progestin; this finding needs to be confirmed and explored further in future studies.