Leptin is a major determinant of energy homeostasis, acting both centrally and in the gastrointestinal tract. We previously reported that acute leptin treatment enhances the absorption of di- and tripeptides via the proton-dependent PepT1 transporter. In this study, we investigated the long term effect of leptin on PepT1 levels and activity in Caco2 cell monolayers in vitro. We then assessed the significance of the regulation of PepT1 in vivo in a model of diet-induced obesity. We demonstrated that 1) leptin regulated PepT1 at the transcriptional level, via the MAPK pathway, and at the translational level, via ribosomal protein S6 activation, in Caco2 cells and 2) this activation was systematically followed by a time- and concentration-dependent loss of leptin action reflecting desensitization. Deciphering this desensitization, we demonstrated that leptin induced a down-regulation of its own receptor protein and mRNA expression. More importantly, we showed, in mice with diet-induced obesity, that a 4-week hypercaloric diet resulted in a 46% decrease in PepT1-specific transport, because of a 30% decrease in PepT1 protein and a 50% decrease in PepT1 mRNA levels. As shown in Caco2 cells, these changes in PepT1 were supported by a parallel 2-fold decrease in leptin receptor expression in mice. Taken together, these results indicate that during induction of obesity, leptin resistance may also occur peripherally in the gastrointestinal tract, disrupting the absorption of oligopeptides and peptidomimetic drugs.