PepT1 is an intestinal epithelial apical membrane transporter that is expressed in the small intestine, with little or no expression in the normal colon. However, we previously demonstrated that colonic PepT1 may be expressed during chronic inflammation. To begin elucidating inflammatory hPepT1 signaling, we herein investigated the long term leptin treatments, on PepT1 expression and activity in Caco2-BBE cells, and began to reveal the involved signaling pathways. We successfully cloned the 723-bp hPepT1 promoter region and identified the human transcription initiation site 86 bp upstream from the translation start site. Leptin treatment dose- and time-dependently increased hPepT1 promoter and transport activities in Caco2-BBE cells, with maximal activity observed in cells treated with 100 nM leptin for 8 h. Under these conditions, we observed 2-fold increases in hPepT1 mRNA and protein expression, as well as increased transport activity. Our molecular analyses of possible signal-transduction pathways revealed that leptin treatment enhanced the intracellular levels of cAMP and phosphorylated cAMP-response element-binding protein (CREB) protein in Caco2-BBE cells, whereas our deletion, mutation, and CDX2 overexpression analyses demonstrated that interaction of the Cdx2 and phosphorylated CREB transcription factors was essential for leptin-induced hPepT1 transcription in Caco2-BBE cells. Our results indicate that leptin, which is increased in inflamed colonic mucosa, triggers colonic expression of hPepT1 via the CREB and Cdx2 transcription factors. These findings provide important new insights into the mechanisms of intestinal inflammation and may suggest new therapeutic modalities in the future.