Cation diffusion facilitator (CDF) transporters are a ubiquitous family of divalent metal-proton antiporters that possess broad metal specificity. In the globally significant bacterial pathogen Streptococcus pneumoniae, the CDF family transporter CzcD contributes to resistance against host-mediated zinc (Zn2+) intoxication via the selective efflux of the metal from the cytoplasm. Further, it has been shown that deletion of czcD can impair virulence in murine models of S. pneumoniae infection. Despite the established importance of CzcD in S. pneumoniae Zn2+ homeostasis, there remains limited understanding of the molecular determinants that define metal selectivity and the mechanistic basis for metal translocation. Here, we investigated the contribution of three conserved metal coordinating sites (A, B, and C) within CzcD that have been defined based on studies of orthologous CDF proteins, most notably Escherichia coli YiiP (FieF). We employed a mutagenesis-based framework to evaluate the contribution of each metal-coordinating site in CzcD to metal ion interaction and transport. To complement this approach, isogenic mutant variants were generated in the serotype 2 S. pneumoniae D39 strain to investigate the impact of specific CzcD site mutations on metal ion accumulation and resistance to metal stress. Collectively, this work advances our understanding of the molecular mechanisms of Zn2+ binding and transport by S. pneumoniae CzcD and expands our knowledge of CDF transporters.