Isoniazid (INH) targets cell wall biosynthesis and is a potent antimycobacterial agent. Elucidating the regulatory networks that govern drug susceptibility in mycobacterial models is fundamental to understanding intrinsic resistance in pathogenic species. The iniBAC operon plays a crucial role in INH tolerance and envelope permeability, yet the transcriptional regulatory mechanisms controlling its expression in response to INH-induced stress remain incompletely understood. The second messenger cyclic di-GMP (c-di-GMP) regulates drug susceptibility in several bacteria, but its downstream receptors and regulatory pathways in mycobacteria have not been explored. Here, we demonstrate that c-di-GMP reduces INH susceptibility via the receptor HpoR. Mechanistically, c-di-GMP alleviates HpoR-mediated repression of the iniBAC operon in a concentration-dependent manner, which decreases envelope permeability and consequently modulates multidrug susceptibility in both M. bovis BCG and M. smegmatis. This regulatory paradigm is likely conserved in pathogenic mycobacteria.