In this study, we tested whether the oral administration of EBHU18, one of the newly synthesized fatty acid bile acid conjugates (FABACs), was able to prevent the development of cholesterol gallstones. In the first study, gallstone-susceptible C57BL/6 mice were fed a lithogenic diet for 8 weeks, and then treated with ursodeoxycholic acid (UDCA, 10 mg kg(-1) day(-1), i.g.), EBHU18-L (0.5 mg kg(-1) day(-1) i.g.), or EBHU18-H (3 mg kg(-1) day(-1) i.g.). In the second study, C57BL/6 mice were fed the lithogenic diet for 8 weeks and then treated with EBHU18 at a daily dose of 3 mg (EBHU18-H) or 0.5 mg (EBHU18-L) or with 10 mg of UDCA for another 8 weeks. Pre-treatment or post-treatment with EBHU18 at 3 mg kg(-1) day(-1) significantly decreased gallstone formation and the fatty liver score. In the first study, EBHU18-H significantly decreased gallstone formation compared with the control animals. The fatty liver score decreased from a mean of 1.6 +/- A 1.02 in the controls to 0.5 +/- A 0.67 in the EBHU18-L group (P < 0.05) and 0.3 +/- A 0.64 in the EBHU18-H group (P < 0.01). In the second study, the drug significantly lowered the gallstone formation rate in the EBHU18-H group. The fatty liver score decreased from a mean of 1.6 +/- A 0.98 in the controls to 0.67 +/- A 0.90 in the EBHU18-L group (P < 0.05) and 0.4 +/- A 0.74 in the EBHU18-H group (P < 0.01). EBHU18 can prevent and reduce gallstone formation and/or fatty liver and can be developed as a potential therapeutic candidate for anti-gallstone therapy.