Burkholderia pseudomallei-a causative agent of melioidosis that is endemic in Southeast Asia and Northern Australia-is a Gram-negative bacterium transmitted to humans via inhalation, inoculation through skin abrasions, and ingestion. Melioidosis causes a range of clinical presentations including skin infection, pneumonia, and septicemia. Despite skin infection being one of the clinical symptoms of melioidosis, the pathogenesis of B. pseudomallei in skin fibroblasts has not yet been elucidated. In this study, we investigated B. pseudomallei pathogenesis in the HFF-1 human skin fibroblasts. On the basis of co-culture assays between different B. pseudomallei clinical strains and the HFF-1 human skin fibroblasts, we found that all B. pseudomallei strains have the ability to mediate invasion, intracellular replication, and multinucleated giant cell (MNGC) formation. Furthermore, all strains showed a significant increase in cytotoxicity in human fibroblasts, which coincides with the augmented expression of matrix metalloproteinase-2. Using B. pseudomallei mutants, we showed that the B. pseudomallei Bsa type III secretion system (T3SS) contributes to skin fibroblast pathogenesis, but O-polysaccharide, capsular polysaccharide, and short-chain dehydrogenase metabolism do not play a role in this process. Taken together, our findings reveal a probable connection for the Bsa T3SS in B. pseudomallei infection of skin fibroblasts, and this may be linked to the pathogenesis of cutaneous melioidosis.