Intestinal ischemic injury damages the epithelial barrier and predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age dependency in intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier compared with older patients. We have developed a pig model that demonstrates age-dependent failure of intestinal barrier restitution in neonatal pigs, which can be rescued by the direct application of juvenile pig mucosal tissue, but the mechanisms of rescue remain undefined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells from recovering ischemic jejunum of juvenile pigs underwent single-cell transcriptomics and the predicted upstream regulator, colony stimulating factor-1 (CSF-1), was interrogated in our model. A subcluster of absorptive enterocytes expressed several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by colony stimulating factor-1 (CSF-1). We validated age-dependent induction of CSF-1 by ischemia and documented that CSF-1 and colony-stimulating factor-1 receptor (CSF1R) co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued neonates. Furthermore, the CSF-1 blockade reduced restitution in vitro, and CSF-1 improved barrier function in injured neonatal pigs in preliminary ex vivo experiments. These studies validate an approach to inform potential novel therapeutic targets, such as CSF-1, to improve outcomes in neonates with intestinal injury in a unique pig model.NEW & NOTEWORTHY These studies validate an approach to identify and predict upstream regulation of restituting epithelium in a unique pig intestinal ischemic injury model. Identification of potential molecular mediators of restitution, such as CSF-1, will inform the development of targeted therapeutic interventions for the medical management of patients with ischemia-mediated intestinal injury.