Cyclooxygenase-2 (COX-2), which is expressed by cholangiocytes in biliary tract disorders, has recently been implicated in biliary tract carcinogenesis. The mechanisms responsible for this COX-2 expression remain unclear. In human diseases, bile contains oxygenated derivatives of cholesterol (oxysterols) which possess diverse biological properties. Therefore, we determined if oxysterols modulate COX-2 expression. The effect of an oxysterol (22(R)-hydroxycholesterol, 22-HC) on COX-2 expression in KMBC cells, a human cholangiocarcinoma cell line, was examined. 22-HC enhanced COX-2 protein expression. This oxysterol activated p42/44 and p38 MAPK, but not JNK 1/2. A p42/44 MAPK inhibitor did not block COX-2 induction, while p38 MAPK inhibitor effectively attenuated COX-2 induction. Although COX-2 mRNA levels were increased by 22-HC, this increase was not transcriptionally regulated, as 22-OH did not increase activity in a COX-2 promoter gene assay. In contrast, COX-2 mRNA stability was augmented by 22-HC treatment, and this effect was reversed by a p38 MAPK inhibitor. In conclusion, the results demonstrate that the oxysterol 22-HC stabilizes COX-2 mRNA via a p38 MAPK-dependent mechanism. This enhanced COX-2 protein expression by oxysterols may participate in the genesis and progression of cholangiocarcinoma.