Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) catalyzes the reaction between the low active 17-ketosteroids and the highly active 17beta-hydroxysteroids. In the present study, we have generated transgenic (TG) mice expressing human (h) HSD17B1 under mouse mammary tumor virus (MMTV) promoter (MMTV-hHSD17B1TG mice). The MMTV-hHSD17B1TG mice were used to characterize HSD17B1 enzyme activity and properties of HSD17B1 inhibitor in vivo. Expression of the transgene was detected by enzyme activity and RT-PCR analysis. Increased HSD17B1 activity in the TG mice was detected in vivo by applying estrone as a substrate via an intravenous injection. The developed enzyme activity measurement was then applied to analyze the efficacy of HSD17B1 inhibitor in vivo. The results indicated that the MMTV-hHSD17B1TG mouse model is a valuable novel tool to test human HSD17B1 inhibition by various compounds in vivo. With the potent hHSD17B1 inhibitor compound tested, at highest an 85% and 33% inhibition of the enzyme activity in males and in females, respectively, was observed.