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The freshwater planarian is a powerful animal model for studying regeneration and stem cell activity in vivo. During regeneration, stem cells (neoblasts in planarian) migrated to the wounding edge to re-build missing parts of the body. However, proteins involved in regulating cell migration during planarian regeneration have not been studied extensively. Here we report two small GTPase genes (Djrho2and Djrho3) of Dugesia japonica(strain Pek-1). In situhybridization results indicated that Djrho2was expressed throughout the body with the exception of the pharynx region while Djrho3was specifically expressed along the gastro-vascular system. Djrho2was largely expressed in neoblasts since its expression was sensitive to X-ray irradiation. In Djrho2-RNAi planarians, smaller anterior blastemas were observed in tail fragments during regeneration. Consistently, defective regeneration of visual nerve was detected by immunostainning with VC-1 antibody. These results suggested that Djrho2is required for proper anterior regeneration in planairan. In contrast, no abnormality was observed after RNAi of Djrho3. We compared protein compositions of control and Djrho2-RNAi planarians using an optimized proteomic approach. Twenty-two up-regulated and 26 de-regulated protein spots were observed in the two-dimensional electrophoresis gels, and 17 proteins were successfully identified by Mass Spectrometry (MS) analysis. Among them, 6 actin-binding or cytoskeleton-related proteins were found de-expressed in Djrho2-RNAi animals, suggesting that abnormal cytoskeleton assembling and cell migration were likely reasons of defected regeneration.