Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.