ABSTRACTDespite a century of investigation,Streptococcus pneumoniaeremains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) ofS. pneumoniaeis a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Throughin vitroassays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated thatin vivoPn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3S. pneumoniae. Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused byS. pneumoniaethrough enzymatic hydrolysis of the CPS.