Currently, there is significant interest in maximizing exposures to rifampin in order to eliminate Mycobacterium tuberculosis (Mtb) “persisters”, however the mechanisms of tolerance to rifampin and other anti-tubercular drugs remain largely undefined. In order to address this significant knowledge gap, we conducted a high-throughput transposon (Tn) mutant screen to identify Mtb genes required for tolerance to rifampin in nutrient-rich broth and in physiologically relevant stress conditions. Mutants containing a Tn insertion in the rv2224c/caeA gene were 294-fold more susceptible to rifampin than was wild-type Mtb. This differential antibiotic susceptibility phenotype was confirmed using a targeted caeA deletion mutant (ΔcaeA) and its complemented strain. We also found that caeA deficiency is associated with increased rifampin accumulation and increased permeability to the polar molecule ethidium bromide in Mtb. Using a combination of targeted and unbiased approaches, the current proposal will test the hypothesis that CaeA, a cell wall-associated carboxylesterase and serine protease, mediates Mtb tolerance to rifampin by modifying Mtb cell wall composition, thereby reducing drug permeability. Our findings are expected to have far-reaching implications for understanding antibiotic tolerance in Mtb and other persistent bacterial infections.