ABSTRACT Functional genomics analysis of Mycobacterium abscessus clinical isolates from chronically infected patients to identify genes under strong evolutionary pressure during lung adaptation identified phoR as one of the most frequently mutated. phoR encodes the histidine kinase (HK) of the two-component regulatory system (TCS) PhoPR. While PhoPR has been extensively studied in Mycobacterium tuberculosis for its role in virulence, little is known about the function of this TCS and the signals governing its activation in M. abscessus . We here show that acidic pH leads to the upregulation of phoP in M. abscessus and that clinically relevant non-synonymous mutations identified in PhoR exacerbate this response. PhoR modulates the ability of its cognate response regulator, PhoP, to autoregulate itself by controlling its dephosphorylation. At low pH, the phosphatase activity of PhoR is reduced and the build-up of phospho-PhoP that ensues results in the upregulation of phoP that accompanies the induction of a defined set of genes, many of which are thought to play a role in virulence and host adaptation. In line with the idea that M. abscessus isolates expressing clinically relevant PhoR variants may be better prepared for survival within the host, we find these strains to be less efficiently internalized by macrophages and to display enhanced intracellular survival. IMPORTANCE Difficult-to-treat pulmonary infections caused by nontuberculous mycobacteria of the Mycobacterium abscessus group have been steadily increasing in the USA and globally. Owing to the relatively recent recognition of M. abscessus as a human pathogen, basic and translational research to address critical gaps in diagnosis, treatment, and prevention of diseases caused by this microorganism has been lagging behind that of the better-known mycobacterial pathogen, Mycobacterium tuberculosis . To begin unraveling the molecular mechanisms of pathogenicity of M. abscessus , we here focus on the study of a two-component regulator known as PhoPR which we found to be under strong evolutionary pressure during human lung infection. We show that PhoPR is activated at acidic pH and serves to regulate a defined set of genes involved in host adaptation. Accordingly, clinical isolates from chronically infected human lungs tend to hyperactivate this regulator enabling M. abscessus to escape macrophage killing.