Acquisition of zinc by Streptococcus pneumoniae is essential for colonization and mediating disease. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcBC, and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that in vivo, AdcA and AdcAII act in a complementary manner during host colonization to facilitate a more efficient infection. We have also identified that AdcAII is reliant upon the pneumococcal histidine triad (Pht) proteins to aid in zinc recruitment. Here we show the hierarchical importance of the five histidine triad motifs in PhtD-facilitated zinc acquisition. By combining zinc uptake assays, transcriptional analyses and assessing growth phenotypes of PhtD histidine triad motif mutants we show that the amino-terminal proximal histidine triad motif (site 1) is the most crucial, while the carboxy-terminal proximal histidine triad (site 5) is the least essential in pneumococcal zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the histidine triad mutants indicated that the first, second and fifth motifs had similar importance in colonization. Collectively, our new insights into the contributions of the individual histidine triad motifs PhtD, and by extension the other Pht proteins, highlights their crucial role in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.