EF4 encoded by lepA gene is an elongation factor related to protein translation. To study the function of EF4 in Mycobacterium tuberculosis (M. tuberculosis), a lepA gene knockout strain was constructed in this study. The whole genome DNA of M. tuberculosis H37Ra was used as a template to amplify the left and right arms of lepA gene respectively by polymerase chain reaction (PCR). The fragments were cloned into p0004S plasmid to construct homologous recombinant p0004S-ΔlepA plasmid. p0004S-ΔlepA plasmid was cloned into phAE159 plasmid by phage packaging in vitro to construct a phAE159-ΔlepA phage packaging plasmid. The phage was amplified in M. smegmatis mc2155 and infected M. tuberculosis for homologous recombination. The positive colonies were selected and the EF4 protein were detected. The PCR results showed that lepA gene in the knockout strain was successfully replaced by the hygromycin resistance gene and the results of Western blotting showed that there was no EF4 expression in the knockout strain, indicating that RaΔlepA strain was successfully constructed. Growth curve analysis showed that lepA knockout in M. tuberculosis had no phenotype change under the normal growth conditions. Comparing to the wild-type strain, colonies of RaΔlepA had yellowish color and thicker protrusions. Stress resistance analysis showed that there was no difference in heat resistance, detergent resistance and oxidant resistance between the wild-type strain and RaΔlepA strain, but RaΔlepA strain had enhanced ability to withstand acidic environment.