Metalloenzymes have critical roles in a wide range of biological processes and are directly involved in many human diseases; hence, they are considered as important targets for therapeutic intervention. The specific characteristics of metal ion(s)-containing active sites make exploitation of metal-binding pharmacophores (MBPs) critical to inhibitor development targeting metalloenzymes. This Perspective focuses on boron-containing MBPs, which display unique binding modes with metalloenzyme active sites, particularly via mimicking native substrates or tetrahedral transition states. The design concepts regarding boron-containing MBPs are highlighted through the case analyses on five distinct classes of clinically relevant nucleophilic metalloenzymes from medicinal chemistry perspectives. The challenges (e.g., selectivity) faced by some boron-containing MBPs and possible strategies (e.g., bioisosteres) for metalloenzyme inhibitor transformation are also discussed.