We studied the expression pattern and the role of CD44 in regulating the malignant behavior of two cholangiocarcinoma (CCA) cell lines which expressed different levels of CD44 using the siRNA technique. KKU-100, the high CD44 expresser, exhibited a high degree of in vitro invasiveness, migration and adhesion to Matrigel compared to HuCCA-1. Silencing of CD44 by siRNA did not have a significant effect on cell proliferation. However, in vitro invasiveness, directional migration (chemotaxis) and adhesion to Matrigel were markedly reduced in both cell lines, although chemokinesis and MMP secretion were variable, demonstrating the distinct functional role and requirement for CD44 in different cellular activities and in different cell types. In addition, immunohistochemical analysis suggested that CD44 may be involved in the differentiation process or tumor progression, depending on the macroscopic type of CCA. Taken together, our data indicate that CD44 is an important requirement for the invasive phenotype of CCA cells, although the role that CD44 plays may vary depending on the CCA type and the cellular activity in which it is engaged.