Background: Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously. Results: We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum. Conclusions: Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.