Mycobacterium tuberculosis causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Aminoglycosides, Amikacin (AK) & Kanamycin (KM) are commonly used in tuberculosis treatment and are drugs of choice especially for category II patients. They inhibit protein synthesis in susceptible bacteria by interacting with several steps of translation. Several explanations have been put forward to explain the mechanism of aminoglycoside resistance but still our knowledge is fragmentary. Rv0148 was found to be overexpressed in AK & KM resistant isolates. To establish the relationship of Rv0148 with AK & KM resistance it was cloned, expressed and antimicrobial drug susceptibility testing (DST) was carried out. Gene was cloned and expressed in E.coli BL21 using pQE2 expression vector. Etest results for drug susceptibility testing against AK & KM showed that the MIC of recombinant cells with Rv0148 was altered. Recombinants showed three fold changes in MIC with AK and two fold with KM E-strips. These MIC shifts speculate, overexpression of Rv0148 protein might be playing a pivotal role in the survival of mycobacteria by inhibiting/modulating the effects of AK & KM.