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Mycobacterium bovis, a member of the M. tuberculosis complex, causes bovine tuberculosis, one of the most important veterinary health problems in the UK. In the absence of improved control the projected economic burden in the UK over the next decade is predicted to be £1bn. Although transmission of M. bovis occurs primarily between infected mammalian hosts, M. bovis has been demonstrated to persist in soil, suggesting an environmental infectious route. M. bovis is likely predated on by environmental amoebae such as Dictyostelium discoideum and as such may have evolved mechanisms to modulate the interaction with amoebae. In this study we have investigated M. bovis interactions in vitro with D. discoideum. We demonstrate that virulent M. bovis evades destruction by D. discoideum. Using a genome-wide M. bovis transposon mutant library, we selected for mutants that failed to escape D. discoideum after 48 h of infection. Mutants of genes encoding the MCE4 transport system, genes involved in sulpholipid synthesis/transport, and genes encoding PPE and PE-PGRS protein families remained associated with D. discoideum. Most strikingly, mutations in 11 genes of the major mycobacterial virulence locus ESX-1, which encodes a T7 secretion system implicated in bacterial transit through host cell membranes, were significantly enriched in D. discoideum. Our data demonstrate that known virulence factors involved in host-pathogen interactions in mammalian hosts also play a role in D. discoideum-M. bovis interactions. Our data further suggest that M. bovis has evolved to actively transit bacteriverous D. discoideum rather that to use it as a replicative niche.