Biliary ducts are lined with epithelial cells, which consist of at least two types of cholangiocytes, small and large. In contrast to large cholangiocytes, which are involved in secretion, the role of small cholangiocytes has not been elucidated. To address this question, we analyzed migration-based characteristics of these cells that may help to understand their functions in vivo. Interestingly, dibutyryl cAMP induced marked filopodia formation and cdc42 activation in NMC-S as compared with NMC-L. Analysis of members of EphA family of guidance molecules revealed a distinct subcellular distribution of EphA5 and EphA8 members: EphA8 was equally expressed by both cell types and localized subcellularly in peripheral cell membranes, while EphA5 was expressed predominantly in NMC-S and localized to filopodia. Moreover cAMP inducible filopodia formation in these cells was abrogated using EphA5 siRNA. Finally, we found that the Rho family GTPase cdc42 was activated in a manner dependent on EphA5. Wortmannin, specific inhibitor of PI3-K abolished the activation of cdc42 dependent on EphA5 suggests the involvement of PI3-K in EphA5-cdc42 pathway. Together, our findings suggests a cAMP-EphA5-cdc42-dependent regulation of small cholangiocyte migration, which are anticipated to facilitate understanding of the nature of cholangiocytes and to explain certain general aspects of cAMP-cdc42 activation signaling with regard to cell morphogenesis.