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<i>Burkholderia pseudomallei</i> is a pathogenic bacterium that causes human melioidosis, which is associated with a high mortality rate. However, the underlying mechanisms of <i>B. pseudomallei</i> pathogenesis are largely unknown. In this study, we examined the infection of human neuronal SH-Sy5y cells by several clinically relevant <i>B. pseudomallei</i> strains. We found that all tested <i>B. pseudomallei</i> strains can invade SH-Sy5y cells, undergo intracellular replication, cause actin-tail formation, and form multinucleated giant cells. Additionally, a deletion mutant of <i>B. pseudomallei</i> cycle-inhibiting factor (<i>cif</i>) was constructed that exhibited reduced invasion in SH-Sy5y cells. Complementation of <i>cif</i> restored invasion of the <i>B. pseudomallei</i> <i>cif</i>-deleted mutant. Our findings enhance understanding of <i>B. pseudomallei</i> pathogenicity in terms of the virulence factor Cif and demonstrate the function of Cif in neurological melioidosis. This may eventually lead to the discovery of novel targets for treatment and a strategy to control the disease.