As a severe adverse effects of afatinib, a tyrosine kinase inhibitor (TKI), diarrheas lead to mortality and morbidity in cancer patients. This study was designed to investigate the effect of afatinib on intestinal epithelial barrier integrity using a human colonoid model. Afatinib at 0.5 µM significantly decreased the transepithelial electrical resistance (TEER) in human colonoids (Mean± S.D.= 100 ± 12.2% vs 32 ± 5.4%; p-value = 0.0003, n =5, one-way ANOVA followed by Tukey test). Delocalization of zonular occluding-1 (ZO-1) and a decrease in mRNA and protein expression of claudin-4 and ZO-1 were observed in the afatinib-treated human colonoids (n =5). Nuclear translocation of nuclear factor kappa B (NF-κB) and mRNA expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) were also found after afatinib treatment (n =5). Importantly, mRNA and protein expression of myosin light chain (MLC) kinase (MLCK) and MLC phosphorylation, a known inducer of intestinal epithelial barrier disruption, were observed (n =5). Treatment with iNOS inhibitor or MLCK inhibitor suppressed the effect of afatinib on TEER (Mean± S.D. = 22 ± 1.2 % vs 36 ± 3.5 % relative to non-treated group, p-value = 0.004, n = 5, one-way ANOVA followed by Tukey test). Collectively, our results indicate that afatinib induces intestinal epithelial barrier dysfunction via NF-κB-iNOS-MLCK-dependent mechanisms. These findings provide insight into pathophysiology of afatinib-induced diarrheas.