Isoniazid (INH) is an antibiotic that is widely used to treat tuberculosis (TB). Adaptation to environmental stress is a survival strategy forMycobacterium tuberculosisand is associated with antibiotic resistance development. Here, mycobacterial adaptation following INH treatment was studied using a multi-stress system (MS), which mimics host-derived stress. Mtb H37Rv (drug-susceptible), mono-isoniazid resistant (INH-R), mono-rifampicin resistant (RIF-R), and multidrug-resistant (MDR) strains were cultivated in the MS with or without INH. The expression of stress-response genes (hspX,tgs1,icl1, andsigE) and lipoarabinomannan (LAM)-related genes (pimB,mptA,mptC,dprE1,dprE2, andembC), which play important roles in the host–pathogen interaction, were measured using real-time PCR. The different adaptations of the drug-resistant (DR) and drug-susceptible (DS) strains were presented in this work.icl1anddprE1were up-regulated in the DR strains in the MS, implying their roles as markers of virulence and potential drug targets. In the presence of INH,hspX,tgs1, andsigEwere up-regulated in the INH-R and RIF-R strains, whileicl1and LAM-related genes were up-regulated in the H37Rv strain. This study demonstrates the complexity of mycobacterial adaptation through stress response regulation and LAM expression in response to INH under the MS, which could potentially be applied for TB treatment and monitoring in the future.