Flagellin acting via TLR5 (Toll-like receptor 5) is a key regulator of the host response to the gut microbial flora in both health and disease. The present study has investigated regulation of flagellin-TLR5 signalling in human colonocytes (HT29-19A) by IFNγ (interferon-γ), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function thatmay facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNγ did not induce chemokine secretion, but markedly potentiated responses to flagellin, increasing CXL8 gene expression and protein secretion by approx. 4-fold. Potentiation by IFNγ was independent of changes in TLR5 and was associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88 (myeloid differentiation factor 88). Knockdown of MyD88 expression using siRNA (small interfering RNA) abolished flagellin-dependent CXCL8 secretion and the potentiating effect of IFNγ. Exposure of non-transformed mouse and human colonocytes to IFNγ also increased MyD88 expression. STAT (signal transducer and activator of transcription) 1 knockdown and use of the broad-spectrum JAK (Janus kinase)-STAT inhibitor AG490 had no effect on IFNγ-mediated up-regulation of MyD88. The findings of the present study suggest that IFNγ sensitizes colonic epithelial cells to bacterial flagellin via a largely STAT-independent up-regulation of MyD88 expression leading to increased secretion of immunomodulatory factors. These results indicate that epithelial responses to flagellin are potentiated by IFNγ, most likely mediated by increased MyD88 expression. The present study adds to our understanding of the spectrum of effects of this cytokine on gut epithelium that may contribute to bacterial-driven inflammation in the gut.