Mouse cytochrome P450 2b9 (Cyp2b9) is a testosterone 16α-hydroxylase enzyme showing female-specific expression in many inbred mouse strains, including C57BL/6J. Previous studies have recognized that some sex-dependently secreted endogenous modulating factors were involved in the sexually dimorphic expression of Cyp2b9 through transcriptional regulation. In this study, we found evidence that some microRNAs contributed to the sexually biased expression of Cyp2b9 via post-transcriptional regulation. Cyp2b9 was upregulated in livers of hepatocyte-specific Dicer1 knockout mice at 3 weeks. The age-dependent downregulation of Cyp2b9 in the livers of male mice was diminished when Dicer1 was specifically knocked out in hepatocytes. When these data were combined with bioinformatics analysis and microRNA profiles of male and female mice, we found that 18 microRNAs were associated with the sexually dimorphic expression of Cyp2b9, which showed higher expression levels in male C57BL/6J mice when compared with females. Luciferase assays revealed that approximate half of these microRNAs repressed luciferase activity in a reporter system containing the 3'-untranslated region (3'-UTR) of Cyp2b9, and also inhibited Cyp2b9 protein expression. MicroRNA seed region mutation or mutations in putative binding sites of the microRNAs in Cyp2b9 3'-UTR led to the loss of the suppression of luciferase activity. There was also a negative correlation between the levels of these microRNAs and Cyp2b9. Our results suggested that multiple microRNAs participated in the regulation of Cyp2b9 expression, and that the lower expression levels of these microRNAs potentially contributed to the female-specific expression of Cyp2b9 in the livers of C57BL/6J mice.