The emergence of multidrug-resistant and extensively drug-resistant strains poses serious challenges to global tuberculosis control, highlighting the urgent need to elucidate the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant (Msm) mutants and identified a strain, Msm-R1-2, exhibiting 16- and 64-fold increases in minimum inhibitory concentrations (MICs) to BTZ and linezolid (LZD), respectively, compared to the parental strain. Whole-genome sequencing revealed resistance-associated mutations in two functionally distinct genes: , encoding a transcriptional regulator involved in efflux pump expression, and , encoding a porin protein. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that single mutations in either or caused low-level resistance (4-fold MIC increase) to BTZ and LZD, while dual mutations conferred resistance levels comparable to Msm-R1-2, with 16- and 64-fold increases in MICs for BTZ and LZD, respectively. An ethidium bromide accumulation assay demonstrated that mutations in reduce cell wall permeability, contributing to multidrug resistance. Furthermore, quantitative real-time PCR showed that mutations in upregulate the - efflux system. Together, these dual mechanisms function synergistically: restricted drug entry combined with enhanced drug efflux confers robust multidrug resistance. These findings provide novel insights into the evolutionary mechanisms of resistance in mycobacteria.