The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+-dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Cα functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+-binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+-independent C2A domain of double C2 protein γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2γ contains six Arg residues (177RLRRRRR183) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2γ. Because of the presence of the basic cluster, the C2A domain of Doc2γ did not show Ca2+-dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.