Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell-cell adhesion is not formed in MDCK cells in which annexin II, a Ca(2+)- and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell-cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell-cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell-cell adhesion. These results indicate that the nectin-based cell-cell adhesion and afadin, but not the cadherin-based cell-cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions.