The interaction of Helicobacter pylori with cancer-associated fibroblasts (CAFs) to promote cholangiocarcinoma (CCA) genesis is unclear. We aimed to demonstrate the effect and mechanism of H. pylori on function of CAFs in vitro as well as the role of melatonin as an anti-fibrotic agent capable of modulating CAFs. CAF cells were generated by co-culture of human fibroblasts (OUMS cell line) with O. viverrini-associated CCA cells (KKU-100 cell line). In the presence of H. pylori lysate, these CAF cells exhibited increased proliferation and migration. The interaction of CAFs and H. pylori lysate also promoted KKU-100 cell migration. Proteomic analysis revealed that the fibrosis-associated integrin signaling pathway was enriched in CAFs stimulated by H. pylori lysate. Expression of focal adhesion kinase (FAK), a molecule that plays a pivotal role in cell proliferation and migration and known to be a downstream target of integrin, was upregulated in CAFs exposed to H. pylori lysate. Interestingly, melatonin treatment significantly attenuated both proliferation and migration of CAFs by reducing FAK phosphorylation and its downstream PI3K and β-catenin. These results suggest that H. pylori promotes proliferation and migration of CAFs cells and possibly fibrosis via the integrin/FAK signaling pathway, which could be attenuated by melatonin treatment.