Monocarboxylate transporter, MCT1 plays an important role in the absorption of short chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 µM, 24h) up-regulates butyrate transport and MCT1 protein expression in human intestinal Caco2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the current study, we showed that PMA (0.1 µM, 24h) increased the MCT1 promoter activity (-871/+91) by ~ 4 fold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1h and persisted till 24h, suggesting that the effects of PMA are due to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site directed mutagenesis studies showed the predominant involvement of potential AP2 binding site in the activation of MCT1 promoter activity by PMA. In addition, over-expression of AP2 in Caco2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.