Background Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae’s virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth.ResultsMutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis.ConclusionsHere we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0881-6) contains supplementary material, which is available to authorized users.