Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease that can result in asymptomatic, chronic, or acute illness. In acute melioidosis, high levels of proinflammatory cytokines and chemokines are found in organs and blood, and neutrophils play a key role in controlling the infection. We showed that B. pseudomallei activates NF-κB via Toll-like receptor (TLR) 2, TLR4, and TLR5 but can also activate NF-κB and induce interleukin (IL)—8 without involving TLRs. TLR-independent activation depends on a functional Bsa type III secretion system (T3SS) and requires internalization.Themitogen-activated protein kinase (MAPK) inhibitors for p38 and c-Jun N-terminal kinase (JNK) severely impaired IL-8 induction by B. pseudomallei and reduced bacterial internalization. Furthermore, the T3SS mutant induced less JNK phosphorylation than did wild-type bacteria. Thus, in cells with no or low expression of TLRs, such as mucosal epithelial cells, B. pseudomallei can induce IL-8 via NF-κB and MAPK pathways, aided by Bsa T3SS.