Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer. Display omitted •miR-143/145 are essential for mouse intestinal epithelial regeneration after injury•miR-143/145 expression and function are restricted to the intestinal mesenchyme•Upregulated IGFBP5 and reduced IGF signaling correlate with regenerative failure•Results challenge a cell-autonomous tumor suppressor role for miR-143/145 in colon miR-143/145 are expressed and function exclusively within the mesenchyme of intestine to regulate proregenerative signaling and wound healing, challenging a long-presumed role for these microRNAs as cell-autonomous tumor suppressors in colon cancer.