Streptococcus pneumoniae(the pneumococcus) is a bacterial pathogen of global significance, responsible for more than 1 million deaths per year. Essential to the pathogenicity of S. pneumoniaeis the efficacious acquisition of transition metal ions at the host-pathogen interface. Accordingly, the mechanisms that facilitate metal ion uptake are potential antimicrobial targets. The type II ATP binding cassette (ABC) transporter PsaBCA is the sole manganese (Mn2+) acquisition pathway in the pneumococcus and is essential for in vivo virulence. The ABC permease complex comprises a homodimer of heterodimers (PsaB2C2), to which Mn2+is delivered by the extra-cytoplasmic protein PsaA. Here, we investigated the structure and activity of PsaBC, and its interaction with PsaA. The structure of PsaBC was determined by X-ray crystallography and refined to 2.85 Å resolution. Assisted by molecular dynamics simulations of membrane-embedded PsaBC, analysis of the structure identified residues potentially involved in the Mn2+translocation mechanism, which include a putative metal ion binding site, translocation gates and docking residues. Comparative sequence analysis revealed that similar residues recur in many transition metal ion ABC permeases and indicates an essential function. The roles of these residues were investigated by alanine substitution into the psaC gene in S. pneumoniae. The competence of these mutant strains was assessed by growth in low Mn2+media and the accumulation of metal ions was measured by inductively coupled plasma-mass spectrometry (ICP-MS). It was found that residues participating in putative binding sites in the internal channel and at the PsaA/PsaC interface are indispensable for accumulation of Mn2+and growth. The crucial role of these conserved residues strongly suggests that PsaBC interacts directly with the metal ion in order to facilitate directional transport through the transmembrane domains; indicating a novel molecular mechanism for solute import distinct from other Type II ABC permeases. This finding resolves a fundamental question as to how bacterial cells import Mn2+ions and provides structural and functional insights essential for future antimicrobial design.