Johne's disease is a chronic granulomatous enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is responsible for considerable economic losses in the livestock industry and in particular within the dairy sector. A more effective vaccine against Johne's disease would be of major benefit. In this study, we developed an efficient procedure for identifying mutants of MAP with reduced virulence that are potential live vaccine candidates against Johne's disease. A mariner transposon was used to create random insertional libraries in two different MAP strains (989 and k10), an effective cattle macrophage survival system was developed, and a total of 1890 insertion mutants were screened by using a 96-prong multi-blot replicator (frogger) system. Two of the transposon mutants with poor survival ability in macrophages were tested in mice. These strains were found to be attenuated in vivo, thereby validating the further use of this macrophage screening system to identify MAP mutants with potential as candidate vaccines against Johne's disease.