Frequent exposure of the intestine to hostile pathogens and harsh toxins demands rapid and seamless repair. Because the intestine is located inside the body cavity, examination of the intestine’s physiological repair processes has traditionally been limited to fixed tissues. Here we use an intravital imaging methodology, Bellymount, to monitor real-time intestinal injury and subsequent repair in individual animals over multiple days. Within 24 hours of toxin feeding, components of the intestinal brush border, such as Moesin::GFP and Meduse::GFP, relocalize from lumenal cell surfaces to lateral cell surfaces; after 48 hours, they disappear entirely. Concomitant with loss of the intestinal brush border, Moesin::GFP and Meduse::GFP appear in nascent brush border membranes associated with new stem cell progeny. Unlike new brush border formation during normal intestinal renewal, which involves a strikingly large, Pre-Assembled Apical Compartment (PAAC) at tri-cellular junctions (Moreno-Roman et al., bioRxiv 457819), new brush border formation during repair involves linear clusters of 4-8 miniature PAACs at bi-cellular junctions. We call these clusters multi-PAACs. Either genetic ablation of the brush border in mature cells or activation of the injury response pathway JAK-STAT in stem cell progeny is sufficient to induce multi-PAACs in otherwise healthy guts. These findings delineate how damage signals trigger an injury-specific morphogenetic process. Together, these results reveal spatiotemporal dynamics of brush border damage and regeneration and provide insights into fundamental mechanisms of epithelial barrier repair.