Interferon-γ (IFNγ) is an important immunoregulatory cytokine that can also decrease intestinal epithelial barrier function. Little is known about the intracellular signalling events immediately subsequent to IFNγ/IFNγ receptor interaction that mediate increases in epithelial permeability; data that could be used to ablate this effect of IFNγ while leaving its immunostimulatory effects intact. This study assessed the potential involvement of Src family kinases in IFNγ-induced increases in epithelial permeability using confluent filter-grown monolayers of the human colon-derived T84 epithelial cell line. Inhibition of Src kinase with the pharmacologic PP1 and use of Fyn kinase-specific siRNA significantly reduced IFNγ-induced increases in epithelial permeability as gauged by translocation of noninvasive E. coli (HB101 strain) and flux of horseradish peroxidase (HRP) across monolayers of T84 cells. However, the drop in transepithelial resistance elicited by IFNγ was not affected by either treatment. Immunoblotting revealed that IFNγ activated the transcription factor STAT5 in T84 cells, and immunoprecipitation studies identified an IFNγ-inducible interaction between STAT5b and the PI3K regulatory subunit p85α through formation of a complex requiring the adaptor molecule Gab2. siRNA targeting STAT5b and Gab2 reduced IFNγ-induced increases in epithelial permeability and phosphorylation of PI3K(p85α). PP1 and Fyn siRNA reduced IFNγ-induced PI3K activity (indicated by decreased phospho-Akt) and the formation of the STAT5b/PI3K(p85α) complex. Collectively, the results suggest the formation of a Fyn-dependent STAT5b/Gab2/PI3K complex that links IFNγ to PI3K signalling and the regulation of macromolecular permeability in a model enteric epithelium.