Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2DeltapotD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2DeltapotD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (alpha-dimethyl-fluoroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.