Cyanobacteria are among the simplest organisms that show daily rhythmicity. Their circadian rhythms consist of the localization, interaction, and accumulation of various proteins, including KaiA, KaiB, KaiC, and SasA. We have determined the 1.9-angstroms resolution crystallographic structure of the cyanobacterial KaiB clock protein from Synechocystis sp. PCC6803. This homotetrameric structure reveals a novel KaiB interface for protein-protein interaction; the protruding hydrophobic helix-turn-helix motif of one subunit fits into a groove between two beta-strands of the adjacent subunit. A cyanobacterial mutant, in which the Asp-Lys salt bridge mediating this tetramer-forming interaction is disrupted by mutation of Asp to Gly, exhibits severely impaired rhythmicity (a short free-running period; approximately 19 h). The KaiB tetramer forms an open square, with positively charged residues around the perimeter. KaiB is localized on the phospholipid-rich membrane and translocates to the cytosol to interact with the other Kai components, KaiA and KaiC. KaiB antagonizes the action of KaiA on KaiC, and shares a sequence-homologous domain with the SasA kinase. Based on our structure, we discuss functional roles for KaiB in the circadian clock.