Rat carboxylesterase 2 (rCES2), which was previously identified as a methylprednisolone 21-hemisuccinate hydrolase, is highly inducible by dexamethasone in the liver. In the present study, we investigated the molecular mechanisms by which this induction occurs. Injection of dexamethasone (1 mg/kg weight) into rats resulted in increases in the expression of rCES2 mRNA in a time-dependent manner with a peak at 12 h after injection. In primary rat hepatocytes, the expression level of rCES2 mRNA was increased by treatment with 100 nM dexamethasone, and the increase was completely blocked in the presence of 10 µM mifepristone (RU-486), a potent inhibitor of glucocorticoid receptor (GR), or 10 µg/mL cycloheximide, a translation inhibitor. Luciferase assays revealed that 100 nM dexamethasone increased rCES2 promoter activities, although the effect of dexamethasone on the promoter activity was smaller than that on rCES2 mRNA expression. The increased activities were completely inhibited by treatment of the hepatocytes with 10 µM RU-486. Based on these results, it is concluded that dexamethasone enhances transcription of the rCES2 gene via GR in the rat liver and that the dexamethasone-mediated induction of rCES2 mRNA may be dependent on de novo protein synthesis. Our results provide clues to understanding what compounds induce rCES2.