Fibroblasts play important roles in several carcinomas. The proliferative effect of secreted substances from cholangiocarcinoma (CCA)-associated fibroblasts has been recently reported. This study aims to investigate the molecular mechansim underlying fibroblast-induced tumorigenic effects. The gene expression profile of fibroblasts isolated from CCA tissues was performed using oligonucleotide microarrays. The differentially expressed genes and the promising roles in cancer promotion and progression were listed. Periostin (PN) was markedly over-expressed in cancer fibroblasts confirmed by real time RT-PCR and western blot analysis. Patients with intrahepatic CCA showed the expression of PN solely in stromal fibroblasts, but was expressed neither in cancer cells nor immune cells. Low to no expression of PN was observed in tissues of benign liver disease and hepatocellular carcinoma. CCA patients with high levels of PN had significantly shorter survival time than those with low levels (P = 0.029). Multivariate analysis revealed high levels of PN (P = 0.039) and tumor staging (P = 0.036) as independent prognostic factors. The in vitro study revealed that recombinant PN induced CCA cell proliferation and invasion. Integrins (ITGs) as receptors of PN were explored their expressions in CCA cell lines and revealed high level of ITGs _5_1 and _6_4 in CCA cells. Cell adhesion assay revealed that cells favored to bind PN with ITG_5_1. Cells with ITG_5_1 knockdown had reduced PN-induced invasion in corresponding to the decreased level of pAKT level. It is suggested that PN-activated CCA cell invasion via ITG_5_1 and AKT-dependent signaling pathway. In conclusion, the gene expression profile of fibroblasts in CCA is apparently explored and has determined the genes involving in cancer progression. Though PN is explored and determined as a poor prognostic factor, other fibroblast-derived proteins are of great interest to explore their tumorigenic roles and mechanisms of action. Regulation of fibroblast-derived substances in CCA progression may be considered as an alternative therapeutic approach.