Cholesterol 25-hydroxylase (CH25H) catalyzes the production of 25-hydroxycholesterol (25-HC), an oxysterol that can play an important role in different biological processes. However, the mechanisms regulating CH25H expression have not been fully elucidated. In this study, we determined that CH25H is highly expressed in mouse liver and peritoneal macrophages. We identified several liver X receptor (LXR) response elements (LXREs) in the human CH25H promoter. In HepG2 cells, activation of LXR by 25-HC or other oxysterols and synthetic ligands [T0901317 (T317) and GW3965] induced CH25H protein expression, which was associated with increased CH25H mRNA expression. 25-HC or T317 activated CH25H transcription in an LXRE-dependent manner. Thus, high-expressing LXRα or LXRβ activated CH25H expression, and the activation was further enhanced by LXR ligands. In contrast, inhibition of LXRα/β expression attenuated 25-HC or T317-induced CH25H expression. Deficiency of interferon γ expression reduced, but did not block, LXR ligand-induced hepatic CH25H expression. Activation of LXR also substantially induced macrophage CH25H expression. In vivo, administration of GW3965 to mice increased CH25H expression in both liver and peritoneal macrophages. Taken together, our study demonstrates that 25-HC can activate CH25H expression in an LXR-dependent manner, which may be an important mechanism to exert the biological actions of 25-HC.