Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, the functions of Pdx1 in mature small intestine are less known. We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated in response to inactivation of Pdx1 specifically in the intestinal epithelium. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Total RNA was isolated from the first 5 cm of the small intestine from mature Pdx1(flox/flox);VilCre and littermate control mice. Microarray analysis identified 86 probe sets representing 68 genes significantly upregulated or downregulated 1.5-fold or greater in Pdx(flox/flox);VilCre mice maintained under standard conditions. Ingenuity Pathway Analysis revealed that functions of the differentially expressed genes are significantly associated with metabolism of nutrients including lipids and iron. Network analysis examining the interactions among the differentially expressed genes further supports the notion that Pdx1 may modulate metabolism of lipids and iron from mature intestinal epithelium. Following forced oil feeding, Pdx1(flox/flox);VilCre mice showed diminished lipid staining in the duodenal epithelium and decreased serum triglyceride levels, indicating reduced lipid absorption compared with control duodenal epithelium. Blood samples from Pdx1(flox/flox);VilCre mice have significantly lower mean values for mean corpuscular volume and mean corpuscular hemoglobin, consistent with iron deficiency. The absence of nonheme iron in the villous epithelium and lamina propria of Pdx1(flox/flox);VilCre duodenum indicates that the duodenal epithelium lacking Pdx1 may have defects in importing iron through enterocytes, resulting in iron deficiency in Pdx1(flox/flox);VilCre mice.