The intestinal epithelial barrier (IEB) plays a key role in the maintenance of gut homeostasis and the development of the immune system in newborns. The enteric nervous system (ENS), a key regulator of gastrointestinal functions, has been shown to be modulated by nutritional factors. However, it remains currently unknown whether maternal diet, in particular n-3 polyunsaturated fatty acids (n-3PUFAs), can impact upon the IEB in newborn piglets and whether the ENS is involved in this effect. Sows received either a control diet (lard based) or an n-3PUFA diet (linseed oil based) during gestation and lactation. Intestinal paracellular permeability was assessed in Ussing chambers on piglets at birth, 3, 7, 14, 21 and 28 postnatal days (PND). Basal jejunal permeability increased significantly and similarly in both groups until PND14 and decreased thereafter. However, at PND28, permeability was higher in n-3PUFA animals as compared to controls. In addition, a vasoactive intestinal peptide (VIP) receptor antagonist increased paracellular permeability in controls but not in n-3PUFA piglets. Conversely, atropine and hexamethonium decreased paracellular permeability in the n-3PUFA group but not in the control group. Moreover, the n-3PUFA diet increased the proportion of choline acetyltransferase (ChAT)-immunoreactive (IR) neurons and decreased the proportion of VIP-IR neurons in the submucosal plexus of piglet jejunum compared to controls. In addition, in primary culture of rat ENS, we showed that 20:5n-3 but not 18:3n-3 increased the proportion of ChAT-IR neurons and decreased the proportion of VIP-IR neurons. In conclusion, supplementation of the maternal diet with n-3PUFAs modified intestinal permeability probably via diet-induced neuroplastic changes in the ENS of newborn piglets.