Cholangiocarcinoma (CC) and hepatocellularcarcinoma (HCC) are two main forms of liver malignancies, which exhibit differences in drug response and prognosis. Immunohistotochemical staining for cytokeratin markers has been used to some success in the differential diagnosis of CC from HCC. However, there remains a need for additional markers for increased sensitivity and specificity of diagnosis. In this study, we have identified a p38 MAP kinase, p38δ (also known as MAPK13 or SAPK4) as a protein that is upregulated in CC relative to HCC and to normal biliary tract tissues. We performed microarray gene expression profiling on 17 cases of CC, 12 cases of adjacent normal liver tissue, and three case of normal bile duct tissue. p38δ was upregulated in 16 out of 17 cases of CC relative to normal tissue. We subsequently performed immunohistochemical staining of p38δ in 54 cases of CC and 54 cases of HCC. p38δ staining distinguished CC from HCC with a sensitivity of 92.6% and a specificity of 90.7%. To explore the possible functional significance of p38δ expression in CC, we examined the effects of overexpression and knockdown of p38δ expression in human CC cell lines. Our results indicate that p38δ is important for motility and invasion of CC cells, suggesting that p38δ may play an important role in CC metastasis. In summary, p38δ may serve as a novel diagnostic marker for CC and may also serve as a new target for molecular based therapy of this disease.