Prohibitin (PHB) is a highly conserved protein that has multiple functions in the cell. We recently demonstrated that PHB plays an important role in combating oxidative stress and its expression is down-regulated in human and animal models of inflammatory bowel disease. Little is known regarding the regulation of PHB expression in intestine or other tissues. In this study we examined the regulation of PHB expression in intestinal epithelial cells using the model cell line Caco2-BBE. We successfully cloned the 1192-bp human PHB promoter region and identified the transcription start site 1594 bp upstream from the translation start site due to an intervening intron. We show that the acute phase cytokine interleukin-6 (IL-6) increases PHB protein and mRNA abundance and induces PHB promoter activation. The IL-6 response element site in the PHB promoter is required for maximal basal promoter activity and responsiveness to IL-6. IL-6 also increases binding of nuclear proteins to the IL-6 response element in the PHB promoter that are supershifted by a STAT3 antibody. Both basal promoter activity and IL-6 responsiveness are attenuated by signal transducer and activator of transcription 3 short interference RNA, suggesting that signal transducer and activator of transcription 3 mediates PHB activity by IL-6. Confirming these in vitro results, IL-6(-/-) mice exhibit reduced PHB expression in the colon compared with wild-type mice. These results suggest that IL-6 modulates PHB expression in cultured intestinal epithelial cells and in the intestine in vivo.