Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKCiota by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a betabetaalpha fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKCiota PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKCiota-ZIP/p62 complex by mutational analysis. Interestingly, in the PKCiota PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motif-presenting surface, suggesting dual roles for the PKCiota PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.