BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFkappaB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFkappaB activation is unknown. RESULTS: Known T3SS3 effectors are not responsible for NFkappaB activation. Furthermore, T3SS3-null mutants are able to activate NFkappaB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFkappaB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. CONCLUSIONS: T3SS3 does not directly activate NFkappaB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFkappaB activation.