Abstract During planarian regeneration, dorsal and ventral tissues adhere to each other as a result of wound closure. To investigate the role of dorsoventral (DV) interaction in regeneration, dorsal and ventral portions were ectopically reoriented relative to each other by grafting. We sucked a small piece of planarian body into a Pasteur pipette, and then grafted the piece to the original position in DV-reversed orientation. 4 days after grafting, a blastema-like white region was formed on the boundary between the host and the graft. The region grew and formed a cup-shaped projection on both the dorsal and ventral sides of the worm after about 1 week. Histological analysis and in situ hybridization using dorsal and ventral markers revealed that the grafted piece maintained its original DV polarity. Analysis using DV boundary markers clearly indicated that a DV axis was newly established within the projection. Chimeric analysis using a strain-specific marker demonstrated that the projection grew on the DV boundary. Further analysis, using a marker specific for neoblasts and differentiating cells, showed that the projection was composed of neoblasts and/or neoblast-derived cells. Thus, ectopic DV interaction can cause blastema formation, outgrowth and establishment of a DV axis. In addition, staining with tissue- and region-specific markers showed that the projection appears to be identical to the structure from the grafted position to the most anterior or posterior tip. These results suggest that DV interaction evoked by wound closure has a important role in the onset of regeneration, especially for blastema formation and establishment of the DV axis during planarian regeneration.