Streptococcus pneumoniae (the pneumococcus) is a significant human pathogen, responsible for a broad range of diseases including pneumonia. Within the pneumococcus, zinc (Zn(II)) is required for host colonization and pathogenesis. To acquire Zn(II), the bacterium utilises an ABC permease, AdcBC, and two Zn(II)-specific solute-binding proteins, AdcA and AdcAII, for transport of the metal ion across the membrane. Under Zn(II)-limiting conditions, the pneumococcus is reliant upon the highly conserved poly-histidine triad (Pht) proteins to aid in delivery of Zn(II) across the cell wall to the ABC transporter. The Pht proteins (PhtA, B, D and E) each feature 5 or 6 histidine triad (HT) motifs (HXXHXH) that are predicted to bind Zn(II). Here, we examined the importance of the individual HT motifs of PhtD in Zn(II) acquisition. Mutants lacking one of the five HT motifs of PhtD in a ΔadcAΔphtABE background were examined under conditions of Zn(II) restriction. The mutant lacking the HT motif closest to the N-terminus was designated ΔHT1, with the mutant lacking the triad at the C-terminus designated ΔHT5. Similar to the ΔadcAΔphtABDE strain, which lacks all the Pht genes, growth of the ΔHT1 mutant was largely inhibited in Zn(II)-limiting media. In contrast, mutation of the other HT motifs had a lesser effect, with the ΔHT5 strain growing similarly to the parental strain. Zinc acquisition was also assessed, with the ΔHT1-4 strains significantly impaired in Zn(II) uptake while the ΔHT5 strain did not have a significant reduction in Zn(II) uptake. Together, these data indicate that HT1, which is closest to the cell membrane, is the most critical HT motif for Zn(II) acquisition, while the triad closest to the external environment (HT5) has little contribution to Zn(II) uptake. Further characterisation is required to reveal the molecular details of Pht-mediated Zn acquisition.