Group A Streptococcus (GAS) translocates across the host epithelial barrier. Streptococcal pyrogenic exotoxin B (SpeB) directly cleaves junctional proteins. The proteolytic efficacy of SpeB allows GAS to translocate across the epithelial barrier. SpeB-mediated dysfunction of the epithelial barrier may have important implications for not only bacterial invasion but also dissemination of other virulence factors throughout intercellular spaces. Group A Streptococcus (GAS) is an important human pathogen that possesses an ability to translocate across the epithelial barrier. In this study, culture supernatants of tested GAS strains showed proteolytic activity against human occludin and E-cadherin. Utilizing various types of protease inhibitors and amino acid sequence analysis, we identified SpeB (streptococcal pyrogenic exotoxin B) as the proteolytic factor that cleaves E-cadherin in the region neighboring the calcium-binding sites within the extracellular domain. The cleaving activities of culture supernatants from several GAS isolates were correlated with the amount of active SpeB, whereas culture supernatants from an speB mutant showed no such activities. Of note, the wild type strain efficiently translocated across the epithelial monolayer along with cleavage of occludin and E-cadherin, whereas deletion of the speB gene compromised those activities. Moreover, destabilization of the junctional proteins was apparently relieved in cells infected with the speB mutant, as compared with those infected with the wild type. Taken together, our findings indicate that the proteolytic efficacy of SpeB in junctional degradation allows GAS to invade deeper into tissues.