Abstract Clinical and epidemiological studies have clearly demonstrated that women are twice as likely as men to form cholesterol gallstones at all ages studied, indicating that estrogen is an important risk factor for cholesterol gallstone disease. Oral contraceptives and conjugated estrogens significantly increase gallstone prevalence in premenopausal and postmenopausal women. Similar lithogenic effects are also found in men with prostate cancer during postoperative estrogen therapy. All these studies indicate that high susceptibility to gallstones in women compared with men is related to differences in how the liver metabolizes cholesterol in response to estrogen. Our published studies have shown that the classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in estrogen-induced gallstones in female mice. The molecular mechanisms underlying the lithogenic role of estrogen in gallstone formation have become more complicated with the identification of the G protein-coupled receptor 30 (GPR30), a novel estrogen receptor. Our genetic analysis has found that Gpr30 is a new gallstone gene, Lith18, in mice. Our published results have established a novel concept that GPR30 is involved in estrogen-dependent lithogenic actions, working independently of ERα, as both GPR30 and ERα can promote the formation of estrogen-induced gallstones through different pathways. However, identifying the lithogenic mechanisms of GPR30 has been a focal point of interest because it remains elusive how GPR30 increases susceptibility to estrogen-induced gallstones at a molecular level. We hypothesize that GPR30 activated by estrogen enhances cholelithogenesis through the epidermal growth factor receptor (EGFR) signaling pathway by disrupting hepatic bile acid metabolism, promoting biliary cholesterol hypersecretion, and impairing gallbladder emptying and refilling. This hypothesis is based on our new preliminary data showing that GPR30 is localized predominantly in the endoplasmic reticulum of hepatocytes, which is completely different from ERα that resides mainly in the nucleus of hepatocytes. We plan to accomplish our goals by pursuing the following three specific aims: First, we will elucidate the mechanisms whereby the activation of GPR30 enhances the bile lithogenicity by inhibiting hepatic bile acid synthesis through the EGFR pathway. Second, we will investigate the mechanisms underlying the critical role of GPR30 in promoting biliary cholesterol hypersecretion. Third, we will explore whether GPR30 impairs gallbladder motility that accounts for rapid growth and agglomeration of solid cholesterol crystals to microlithiasis. After completing the proposed studies, our results will present a new view on how GPR30 regulates cholesterol and bile acid metabolism in the liver, bile, and gallbladder, and will develop novel concepts to elucidate the vital roles of GPR30 in driving the initiation of supersaturated bile and cholesterol crystallization...