Formation of biofilms by pathogenic bacteria plays a key role in their pathogenesis, especially when the bacteria establish an infection in adverse environments. We examined the genetic basis of biofilm formation in Mycobacterium avium subspecies paratuberculosis(M. ap), the causative agent of Johne's disease in cattle and a potential risk factor associated with Crohn's disease in humans. A transposon mutant of M. ap with an inactivation of the pstA gene was shown to have reduced abilities to form biofilms on PVC plates, and to colonize mouse organs in a murine model of paratuberculosis. Similar results were obtained when a surgical model of intestinal invasion in cattle was utilized to assay the invasion of the pstA mutant, suggesting a role for this gene in biofilm formation and virulence of M. ap. Finally, genome-wide transcriptional analysis of biofilm and planktonic cultures of M. ap profiled M. ap biofilms as stress-responsive structures, especially against oxidation and hypoxia. Overall, the analysis of M. ap biofilm reveals the importance of the pstA gene in biofilm formation and the pathogenesis of M. ap. The knowledge generated in this study will facilitate the analysis of other mycobacterial species that infect humans and animals and can provide a model for the analysis of other biofilm-forming pathogens.