Johne’s disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a major worldwide health problem of domestic and wild animals. All control programmes developed until now against this large epizootie have failed due to the lack of sensitive and specific diagnostic assays and to the lack of an efficient vaccine [1]. The research of new cell wall antigens represent an original alternative to protein antigens typically used in commercial sero-diagnostic tests. Many non-tuberculous mycobacteria such as Mycobacterium avium subsp. avium (Mav) synthesize abundant glycopeptidolipids (GPLs). These surfacelocated GPLs are involved in pathogenicity by interfering with the host immune system. In Mav, GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes [2]. Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. To circumvent the problems of challenging native purification, L5P was chemically synthesized which allows the large-scale production of pure L5P [3]. We further showed that L5P is the target for a highly specific humoral response involving IgM, IgG1 and IgG2 antibodies in Map-infected animals, and that the major epitopes of the L5P are localized in the peptidyl moiety of the molecule. We also showed L5P is the target for a specific humoral response in a subset of human patients with Crohn disease (CD). The L5P, a molecular signature of Map, will open the way to an innovative ELISA diagnosis based on a novel synthetic antigen to unambiguously distinguish Map from Mav, M. bovis or other environmental mycobacteria in sera from livestock or from CD patients [4]. T cell assay and direct detection using specific monoclonal antibodies available should complete diagnosis of Map from blood, milk, and lesions.