The mouse carboxylesterase 2 isozyme, mCES2, is thought to play important roles in lipid metabolism and is expressed in the liver, kidney, and small intestine at high levels. In this study, we examined the molecular mechanisms controlling this tissue-specific expression of mCES2, and demonstrated that hepatocyte nuclear factor-4alpha (HNF-4alpha) could enhance transcription of the mCES2 gene in vitro and in vivo. It was found that effects of HNF-4alpha on the level of mCES2 promoter activity were repressed by small heterodimer partner (SHP) and chenodeoxycholic acid (CDCA) in luciferase assays. Accordingly, mCES2 gene transcription was repressed by CDCA treatment in mouse immortalized hepatocytes. Our results suggested that this repression resulted from the combined effects of both inhibition of HNF-4alpha transactivation ability by SHP and reduction of HNF-4alpha expression level. These findings show that HNF-4alpha plays an important role in the regulation of mCES2 gene transcription.