The small intestine regulates barrier function to absorb nutrients while avoiding the entry of potentially harmful substances or bacteria. Barrier function is dynamically regulated in part by the enteric nervous system (ENS). The role of the ENS in regulating barrier function in response to luminal nutrients is not well understood. We hypothesize that the ENS regulates intestinal permeability and ion flux in the small intestine in response to luminal nutrients. Segments of jejunum and ileum from mice were mounted in Ussing chambers. Transepithelial electrical resistance (TER), short-circuit current (Isc), and permeability to 4-kDa FITC-dextran (FD4) were recorded after mucosal stimulation with either glucose, fructose, glutamine (10 mM), or 5% Intralipid. Mucosal lipopolysaccharide (1 mg/mL) was also studied. Enteric neurons were inhibited with tetrodotoxin (TTX; 0.5 μM) or activated with veratridine (10 μM). Enteric glia were inhibited with the connexin-43 blocker Gap26 (20 μM). Glucose, glutamine, Intralipid, and veratridine acutely modified Isc in the jejunum and ileum, but the effect of nutrients on Isc was insensitive to TTX. TTX, Gap26, and veratridine treatment did not affect baseline TER or permeability. Intralipid acutely decreased permeability to FD4, while LPS increased it. TTX pretreatment abolished the effect of Intralipid and exacerbated the LPS-induced increase in permeability. Luminal nutrients and enteric nerve activity both affect ion flux in the mouse small intestine acutely but independently of each other. Neither neuronal nor glial activity is required for the maintenance of baseline intestinal permeability; however, neuronal activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide.NEW & NOTEWORTHY Luminal nutrients and enteric nerve activity both affect ion transport in the mouse small intestine acutely, but independently of each other. Activation or inhibition of the enteric neurons does not affect intestinal permeability, but enteric neural activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide. The enteric nervous system regulates epithelial homeostasis in the small intestine in a time-dependent, region- and stimulus-specific manner.