[Objective] In order to predict the function of MSMEG_6281 in Mycobacterium smegmatis mc2155, we analyzed the effect of overexpression of MSMEG_6281 on cellular growth and morphological characteristics. [Methods] Ethambutol, one of the first anti-tuberculosis drugs, can destroy structure of cell wall of mc2155 and cause the changes of cellular morphology. The expression change of MSMEG_6281 is determined after ethambutol treatment by RT-PCR. The intact MSMEG_6281 gene was amplified from M. smegmatis mc2155 genomic DNA . After analyzing by enzymatic digestion and DNA sequencing, the MSMEG_6281 gene was cloned into vector pVV16 to constitute the recombinant plasmid of pVV16-MSMEG_6281. The expression of MSMEG_6281 for the recombinant mc2155 strain was confirmed by SDS-PAGE and western blotting. [Results] RT-PCR results show that gene expression of MSMEG_6281 was up-regulated under the condition of ethambutol treatment for 6 h and 12 h. The recombinant mc2155 strain over-expressing MSMEG_6281 demonstrated a lag growth, and the cellular morphological turns longer than wild type mc2155. [Conclusion] MSMEG_6281 must be related with peptidoglycan metabolism of the cell wall.