LytR-CpsA-Psr family proteins play an important role in bacterial cell wall integrity. Although the pathogenic relevance of LytR-CpsA-Psr family proteins has been studied in a few bacterial pathogens, their function in mycobacteria remains uncharacterized. In this work, a transposon insertion mutant (cpsA::Tn) of Mycobacterium marinum was studied. We found that inactivation of CpsA altered bacterial colony morphology, sliding motility, cell surface hydrophobicity, and cell wall permeability. Besides, the cpsA mutant exhibited a decreased arabinogalactan content, indicating that CpsA plays a role in cell wall assembly. Moreover, the mutant shows impaired growth within macrophage cell lines and is severely attenuated in zebrafish larvae and adult zebrafish. Taken together, our results indicated that CpsA, a previously uncharacterized protein, is important for mycobacterial cell wall integrity and is required for mycobacterial virulence.