The genome of Vibrio cholerae contains five flagellin genes that encode proteins (FlaA-E) of 39 to 41-kDa with 61-82% identity among them. Although the existing live oral attenuated, vaccine strains against cholera are protective in humans, there is an intrinsic residual cytotoxic and inflammatory component associated with these candidate vaccine strains. Bacterial flagellins are known to be potent inducers of proinflammatory molecules via activation of Toll-like receptor 5. Here, we found that purified flagella from wild type V. cholerae 395 induced significant release of IL-8 from cultured HT-29 human colonic epithelial cells. Furthermore, we found that filtered supernatants of KKV90, a flaA isogenic mutant unable to produce flagella, were still able to activate production of IL-8, albeit to significantly lower levels than the wild type, suggesting that other activators of proinflammatory molecules were still present in these supernatants. A comparative proteomics analysis of secreted proteins of V. cholerae 395 and KKV90 identified additional proteins with potential to induce IL-8 release in HT-29 cells. Secreted proteins in the range of 30-45-kDa identified by 2-dimensional electrophoresis and mass spectrometry revealed the presence of two additional flagellins FlaC and FlaD, which appeared to be secreted 3- and 6-fold more in the mutant compared to the wild type. Double isogenic mutants flaAflaC and flaAflaD were unable to trigger IL-8 release from HT-29 cells. In sum, we have shown that purified flagella and secreted flagellin proteins (FlaC and FlaD) are inducers of IL-8 release from epithelial cells via Toll-like receptor 5. This observation may explain, in part, the observed reactogenicity of cholera vaccine strains in humans.