Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the β-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining and β-galactosidase immunohistochemistry of fetal tissues. Expression of β-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia.