Ecological functions of cyanophages in aquatic environments depend on their interactions with cyanobacterial hosts. The first step of phage-host interaction involves adsorption to the cell surface. We report that adsorption of a cyanophage, A-1(L), to the outer membrane of Anabaena sp. strain PCC 7120 is based on the binding of a tail protein, ORF36, to the O antigen of lipopolysaccharides (LPS). Removal of O antigen by gene inactivation abolished infection by A-1(L); consistently, preincubation of the cyanophage with extracted Anabaena LPS partially blocked infection. In contrast, inactivation of major outer membrane protein genes in Anabaena or addition of Synechocystis LPS showed no effect on infection. ORF35 and ORF36 are two predicted tail proteins of A-1(L). Antibodies against either ORF35 or ORF36 strongly inhibited infection. Enzyme-linked immunosorbent assay showed a specific interaction between ORF36 and the LPS of Anabaena sp. strain PCC 7120. These findings indicate that ORF35 and ORF36 are probably both required for adsorption of A-1(L) to the cell surface, but ORF36 specifically binds to the O antigen of LPS.IMPORTANCE Cyanophages play an important role in regulating the dynamics of cyanobacterial communities in aquatic environments. Hitherto, the mechanisms for cyanophage infection have been barely investigated. In this study, the first cyanophage tail protein that binds to the receptor (LPS) on cell surface was identified and shown to be essential for the A-1(L) infection of Anabaena sp. strain PCC 7120. The protein-LPS interaction may represent an important route for adsorption of cyanophages to their hosts.