Cholangiocarcinoma (CCA) is a bile duct cancer, commonly found in Asia including Thailand and especially in the northeastern region of Thailand. To identify the proteins involved in carcinogenesis and metastasis of CCA, protein expression profiles of high-invasive KKU-M213 and low-invasive KKU-100 cell lines were compared using a comparative GeLC-MS/MS proteomics analysis. A total of 651 differentially expressed proteins were detected of which 27 protein candidates were identified as having functions involved in cell motility. A total of 22 proteins were significantly upregulated in KKU-M213, whereas 5 proteins were downregulated in KKU-M213. S100A2, a calcium-binding protein in S100 protein family, is upregulated in KKU-M213. S100A2 is implicated in metastasis development in several cancers. The protein expression level of S100A2 was verified by Western blot analysis. Intriguingly, high-invasive KKU-M213 cells showed higher expression of S100A2 than KKU-100 cells, consistent with proteomic data, suggesting that S100A2 may be a key protein involved in the progression of CCA. However, the biological function of S100A2 in cholangiocarcinoma remains to be elucidated. S100A2 might be a potential biomarker as well as a novel therapeutic target in CCA metastasis.