Although perturbations in mitochondrial function and structure have been described in the intestinal epithelium of Crohn's disease and ulcerative colitis patients, the role of epithelial mitochondrial stress in the pathophysiology of inflammatory bowel diseases (IBD) is not well elucidated. Prohibitin 1 (PHB1), a major component protein of the inner mitochondrial membrane crucial for optimal respiratory chain assembly and function, is decreased during IBD.Male and female mice with inducible intestinal epithelial cell deletion of Phb1 (Phb1iIEC ) or Paneth cell-specific deletion of Phb1 (Phb1PC ) and Phb1fl/fl control mice were housed up to 20 weeks to characterise the impact of PHB1 deletion on intestinal homeostasis. To suppress mitochondrial reactive oxygen species, a mitochondrial-targeted antioxidant, Mito-Tempo, was administered. To examine epithelial cell-intrinsic responses, intestinal enteroids were generated from crypts of Phb1iIEC or Phb1PC mice.Phb1iIEC mice exhibited spontaneous ileal inflammation that was preceded by mitochondrial dysfunction in all IECs and early abnormalities in Paneth cells. Mito-Tempo ameliorated mitochondrial dysfunction, Paneth cell abnormalities and ileitis in Phb1iIEC ileum. Deletion of Phb1 specifically in Paneth cells (Phb1PC ) was sufficient to cause ileitis. Intestinal enteroids generated from crypts of Phb1iIEC or Phb1PC mice exhibited decreased viability and Paneth cell defects that were improved by Mito-Tempo.Our results identify Paneth cells as highly susceptible to mitochondrial dysfunction and central to the pathogenesis of ileitis, with translational implications for the subset of Crohn's disease patients exhibiting Paneth cell defects.