Recent studies demonstrate that the structure of ribosome was committed to dynamic regulation during cell development and stress adaptation. We used NCBI BLAST to analyze the sequence similarity of ribosomal protein RpsI, RpmI and RpmJ between Mycobacterium tuberculosis and Mycobacterium smegmatis and found a striking variation in RpsI N terminal. To further study the influence of this variation on ribosomal function, we constructed a M. smegmatis recombinant strain in which the original rpsI was replaced by the counterpart from M. tuberculosis. The genotype of this strain was verified by PCR. We estimated the growth rate of recombinant strain and empty-control strain under different temperatures. Universal serial liquid dilution method was applied to measure the minimum inhibition concentration (MIC₉₀) of five different ribosome-targeting antibiotics. We found that the recombinant strain became more sensitive to amikacin which binds to the A site of ribosome. These results demonstrate that the sequence variation in Mycobacterial RpsI may impact the structure of ribosomal A site.