Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The antimicrobial activity of human β defensin‐1 (hBD‐1) is potentiated by this low‐oxygen environment and the hBD‐1 gene (DEFB1) is constitutively expressed in intestinal epithelial cells (IEC). As previous studies have indicated that hypoxia‐inducible factor (HIF) is active even in healthy intestinal mucosa, we compared the transcriptional profile of IEC in mice lacking IEC HIF‐1α to those of wild‐type mice. This unbiased screen identified a key role for epithelial HIF‐1α in expression of murine defensins. Studies with human IEC revealed that basal HIF‐1α is fundamental for the constitutive expression of hBD‐1 mRNA and protein. While hypoxia did not induce DEFB1 expression, attenuated basal HIF‐1α expression (ascorbate supplementation) repressed DEFB1, suggesting a low threshold for HIF‐1α saturation. DEFB1 promoter studies confirmed HIF‐1α binding to a hypoxia response element. Since DEFB1 is unique to humans, we used 94 human intestinal samples to reveal a strong correlation between DEFB1 and the canonical HIF‐1α target gene GLUT1. Taken together, these findings indicate that basal HIF‐1α is critical for constitutive expression of DEFB1 in the intestine and supports efforts that target HIF in restoration and maintenance of intestinal integrity.Research Support:F30DK096709, TL1RR025778, DK50189, HL60569, DK095491 and by the Crohn's and Colitis Foundation of America.