Burkholderia pseudomallei is an agent of melioidosis and is closely related to avirulent B. thailandensis. Burkholderia thailandensis has a 15-bp deletion within the variable region of the flagellin gene fliC compared with B. pseudomallei. The difference in the fliC gene might be related to virulence. In the present study, the invasion, internalization and intracellular replication of both phagocytic (mouse macrophage cell line RAW264.7) and non-phagocytic cells (human lung epithelial cell line A549) of B. pseudomallei fliC knockout mutant (MM35) complemented with its own fliC (Cp) or with B. thailandensis fliC (Ct) was compared with those of the wild-type strains of B. pseudomallei (1026b) and B. thailandensis (E257). In phagocytic cells, there was no significant difference in bacterial uptake between Cp and Ct, but MM35 was internalized significantly less compared with 1026b, Cp, Ct and E257. The results suggest that flagella are involved in macrophage invasion. In non-phagocytic cells, Cp and Ct showed similar invasive capacities while 1026b, Cp and Ct showed significantly higher invasiveness than MM35, suggesting that flagella facilitate the non-phagocytic cell invasion. However, the invasive capacity of MM35 was significantly higher than that of E257, suggesting that in addition to the flagella, B. pseudomallei may need other factor(s) to facilitate invasion in non-phagocytic cells.