Periostin (PN) (also known as osteoblast-specific factor OSF-2) is a protein that in humans is encoded by the POSTN gene and has been correlated with a reduced survival of cholangiocarcinoma (CCA) patients, with the well-known effect of inducing epithelial-to-mesenchymal transition (EMT). The present study investigated the effect of PN, through integrin (ITG)α5β1, in EMT-mediated CCA aggressiveness. The alterations in EMT-related gene and protein expression were investigated by real-time PCR, western blot analysis and zymogram. The effects of PN on migration and the level of TWIST-2 were assessed in CCA cells with and without siITGα5 transfection. PN was found to induce CCA cell migration and EMT features, including increments in Twist-related protein 2 (TWIST-2), zinc finger protein SNAI1 (SNAIL-1), α-smooth muscle actin (ASMA), vimentin (VIM) and matrix metallopeptidase 9 (MMP-9), and a reduction in cytokeratin 19 (CK-19) together with cytoplasmic translocation of E-cadherin (CDH-1). Additionally, PN markedly induced MMP-9 activity. TWIST-2 was significantly induced in PN-treated CCA cells; this effect was attenuated in the ITGα5β1-knockdown cells and corresponded to reduced migration of the cancer cells. These results indicated that PN induced CCA migration through ITGα5β1/TWIST-2-mediated EMT. Moreover, clinical samples from CCA patients showed that higher levels of TWIST-2 were significantly correlated with shorter survival time. In conclusion, the ITGα5β1-mediated TWIST-2 signaling pathway regulates PN-induced EMT in CCA progression, and TWIST-2 is a prognostic marker of poor survival in CCA patients.