Extracellular vesicles (EVs) have recently garnered attention for their participation in host-microbe interactions in Streptococcus pneumoniae infections. However, the effect of pEVs on the disruption of alveolar epithelial barrier remain poorly understood. Our studies focus on EVs produced by Streptococcus pneumoniae (pEVs), and reveal that pEVs are internalized by alveolar epithelial cells. In vitro, pEVs induce autophagy activation in a dosage-dependent manner and decrease the alveolar epithelial barrier’s trans-epithelium electrical resistance (TEER). In addition, pEV-containing bacterial peotein serine/threonine-protein kinase StkP may act as an activator for Streptococcus pneumoniae-induced autophagy activation. When administered systemically in mice, Streptococcus pneumoniae wild type strain induced acute lung injury, the deletion of stkP deletion strain attenuated this injury. Taken together, pEVs cargos emerge as critical contributors to tissue damage in mammalian hosts.