Listeria monocytogenes is a facultative intracellular pathogen invading humans and animals with the highest fatality rate among the food-borne pathogens. The Listeria pathogenic processes, such as cell entry and escape from phagosomes, depend on the actions of diverse bacterial factors, including lipoproteins. Here, we report the crystal structure of Lmo2642, a conserved putative lipoprotein containing a Ser/Thr phosphatase domain. The protein consists of two distinct domains: a catalytic domain that belongs to the metallophosphoesterase superfamily and an auxiliary α-helical bundle domain. The active site in the catalytic domain of Lmo2642 contains a dinuclear metal center in which Mn2+ and Fe3+ are preferentially positioned at the site1 and site2, respectively. On the basis of the structural analysis and enzymatic assays, we identified the biochemical activity of the protein as a cyclic nucleotide phosphodiesterase toward 2′,3′- and 3′,5′-cyclic nucleotides. Considering the cNMP phosphodiesterase activity and the putative surface localization of Lmo2642, we speculate that Lmo2642 has some potential roles in the host-pathogen interactions by changing the cAMP concentration of host cells during L. monocytogenes infection. Proteins 2011. © 2011 Wiley-Liss, Inc.