Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human β defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.