Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the influences of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut. Display omitted •The mammalian colon exists in a state of relative hypoxia•Hypoxic regions of the normal colon provide a signaling axis through HIF-1•Microbial-derived butyrate depletes O2 and activates HIF-1•Microbiota-derived butyrate is barrier-protective in the mucosa Decreased short-chain fatty acid (SCFA) production by the microbiota has been correlated with colonic inflammation and disease. Kelly et al. show that microbial-derived SCFAs, particularly butyrate, stimulate epithelial metabolism and deplete intracellular O2, resulting in stabilization of the transcription factor HIF-1 and increased epithelial barrier function.