Lipoproteins of temperate phage are a broad family of membrane proteins encoded in the lysogeny module of temperate phages. Expression of theltpTP-J34 gene of temperateStreptococcus thermophilusphage TP-J34 interferes with phage infection at the stage of triggering DNA release and injection into the cell. Here, we report the first structure of a superinfection exclusion protein. We have expressed and determined the X-ray structure of LtpTP-J34. The soluble domain of LtpTP-J34 is composed of a tandem of three-helix helix–turn–helix (HTH) domains exhibiting a highly negatively charged surface. By isolating mutants of lactococcal phage P008wt with reduced sensitivities to LtpTP-J34 and by genome sequencing of such mutants we obtained evidence supporting the notion that LtpTP-J34 targets the phage's tape measure protein (TMP) and blocks its insertion into the cytoplasmic membrane.