Short bowel syndrome is a life-threatening condition with few solutions. Tissue-engineered intestine (TEI) is a potential treatment, but donor intestine is a limiting factor. Expanded epithelial surrogates termed enteroids may serve as a potential donor source.To produce TEI from enteroids, crypts were harvested from mice and enteroid cultures established. Enteroids were seeded onto polymer scaffolds using Matrigel or culture medium and implanted in immunosuppressed mice for 4 wk. Histology was analyzed using Periodic acid-Schiff staining and immunofluorescence. Neomucosa was quantified using ImageJ software. To determine whether TEI could be produced from enteroids established from small intestinal biopsies, 2 × 2-mm pieces of jejunum were processed for enteroid culture, enteroids were expanded and seeded onto scaffolds, and scaffolds implanted for 4 wk.Enteroids in Matrigel produced TEI in 15 of 15 scaffolds, whereas enteroids in medium produced TEI in 9 of 15 scaffolds. Use of Matrigel led to more neomucosal surface area compared to media (10,520 ± 2905 μm versus 450 ± 127 μm, P < 0.05). Histologic examination confirmed the presence of crypts and blunted villi, normal intestinal epithelial lineages, intestinal subepithelial myofibroblasts, and smooth muscle cells. Crypts obtained from biopsies produced an average of 192 ± 71 enteroids. A single passage produced 685 ± 58 enteroids, which was adequate for scaffold seeding. TEI was produced in 8 of 9 scaffolds seeded with expanded enteroids.Enteroids can be obtained from minimal starting material, expanded ex vivo, and implanted to produce TEI. This method shows promise as a solution to the limited donor intestine available for TEI production in patients with short bowel syndrome.