[Background] Burkholderia pseudomallei is an intracellular parasitic bacterium that can cause human disease and even death. The type III secretory system plays an important role in the bacterial invasion of epithelial cells, escaping host immunity and the secretion of virulence factors. The bopA gene is an important effector protein encoded by the TTSS-3 gene, and it plays an important role in the immune escape of the Burkholderia pseudomallei. [Objective] To construct Burkholderia pseudomallei bopA gene knockout mutant strain, and evaluate the biological characteristics of the mutant strain. [Methods] We constructed suicide plasmid pK18mobSacB-ΔbopA, and then transformed the plasmid into B. pseudomallei from Escherichia coli S17-1λpair by conjugation. bopA gene was knocked out by homologous recombination, and the mutant strain was selected by sucrose agar screening. The phenotypic variation of the mutant strain was finally evaluated at the cell and animal levels. [Results] We constructed B. pseudomallei bopA mutant strain successfully, and found the invasion rate, intracellular survival ability as well as the ability of colonization in vivo of mutant strain was significantly reduced. [Conclusion] We constructed B. pseudomallei bopA mutant strain by homologous recombination, to provide a basis for further understanding of this gene.