Streptococcus pneumoniae (pneumococcus) is responsible for significant morbidity and mortality in worldwide. After introduction of current pneumococcal vaccines, a marked decrease in the incidence of pneumococcal disease was observed. Unfortunately, serotype shifts in carriage and disease, including capsular switch and presence of antimicrobial resistance, have been found. Here we report live attenuated vaccine strain which is avirulent and can protect from systemic and mucosal pneumococcal diseases. Pep27, an autolysis-inducing factor of S. pneumoniae is known to mediate LytA-dependent and -independent lysis and it was thus expected to effect virulence. The loss of Pep27 had a much larger than expected decrease in virulence and has made the Pep27 mutant strain sufficiently avirulent to be used as a live vaccine. The pep27 mutation unexpectedly had lower level of capsular polysaccharide than the wild type (type 2, D39) strain. Moreover, the pep27 mutant showed rapid clearance by 24h post intranasal infection, and was not detected in lung and blood suggesting that mutant could not invade into the tissue. Even when 2×10 8CFU were injected intravenously the mutant was not detected in the blood or brain after 4h. Whereas 4h after injection of 6×10 6 CFU of the wild type parent D39 strain, bacteremia was readily detected. Two dose intranasal immunizations with the live pep27 mutant in the absence of adjuvant elicited IgG antibody and serotype-independent protection against lethal intranasal challenge. Thus Pep27 was essential for virulence, and intranasal immunization with the pep27 mutant could provide protective immunity. © 2011 Elsevier Ltd.