Interventricular septum (IVS) formation is one of the key events in the development of a four-chambered heart. We previously showed that the basic helix-loop-helix transcription factor Hand1 plays an important role in the formation of the IVS. Here, we found that the other Hand gene, Hand2, regulated expansion, trabeculation, and IVS formation in the embryonic heart. In transgenic embryos expressing Hand2 in the whole ventricles, the boundary region between the left and right ventricles expanded outwards, resulting in complete absence of the IVS. Moreover, trabecular formation was observed even in a region where the IVS was expected to form. In some transgenic embryos with heterogeneous expression of the transgene, a muscular septum did not form in a region where Hand2 was expressed, but an incomplete septum was identifiable in a region where Hand2 was not expressed, suggesting that septum formation was strictly regulated by the expression domain of Hand2. Furthermore, expression of trabecular markers including ANF, BNP, and connexin40 was significantly up-regulated in the ventricles of Hand2 transgenic embryos as well as in H9c2 cells over-expressing Hand2. These results suggested that the absence of Hand2 expression in the interventricular boundary region inhibits expansion and trabeculation in this area, contributing to the proper formation of the IVS.