Pathogenic mycobacteria have evolved complex metabolic networks that enable persistence and survival in hostile host environments such as phagosomes and granulomas. Among these adaptations, riboswitch-mediated regulation has emerged as a key mechanism for integrating environmental signals with metabolic responses. We are investigating how a glycine-responsive riboswitch controls metabolic and transcriptional regulation that contributes to mycobacterial persistence and virulence. Comparative genomics revealed that pathogenic mycobacteria possess an additional serine hydroxymethyltransferase gene, glyA2, positioned downstream of a glycine riboswitch and upstream of sdaA, thereby forming a regulatory unit that links serine–glycine–folate metabolism to redox balance and DNA synthesis. This genetic configuration is absent from most environmental species, suggesting a role in adaptation to host-derived stress. We hypothesise that glyA2 expression, under riboswitch control, enables detoxification of glycine generated during the breakdown of extracellular matrix components—a process intensified in necrotic lesions. Our multidisciplinary project combines genetics, RNA structural biology, transcriptomics, and infection models to define how ligand binding influences RNA conformation and transcriptional termination, and how these regulatory effects shape cellular responses to glycine. Current work focuses on generating targeted mutants, mapping riboswitch structure–function relationships, and profiling transcriptional and metabolic responses under controlled conditions to determine how this system contributes to survival within host environments. Together, our findings identify glycine detoxification as a bona fide metabolic adaptation specific to pathogenic mycobacteria. Understanding this system offers new insights into host–pathogen interactions and may inform future strategies that exploit metabolic vulnerabilities to enhance antimycobacterial therapy.