Abiogenic lead (Pb²⁺), present in the environment in elevated levels due to human activities, has detrimental effects on human health. Metal-binding sites in proteins have been identified as primary targets for lead substitution resulting in malfunction of the host protein. Although Pb²⁺ is known to be a potent competitor of Ca²⁺ in protein binding sites, why/how Pb²⁺ can compete with Ca²⁺ in proteins remains unclear, raising multiple outstanding questions, including the following: (1) What are the physicochemical factors governing the competition between Pb²⁺ and Ca²⁺? (2) Which Ca²⁺-binding sites in terms of the structure, composition, overall charge, flexibility, and solvent exposure are the most likely targets for Pb²⁺ attack? Using density functional theory combined with polarizable continuum model calculations, we address these questions by studying the thermodynamic outcome of the competition between Pb²⁺ and Ca²⁺ in various model Ca²⁺-binding sites, including those modeling voltage-gated calcium channel selectivity filters and EF-hand and non-EF-hand Ca²⁺-binding sites. The results, which are in good agreement with experiment, reveal that the metal site’s flexibility and number of amino acid ligands dictate the outcome of the competition between Pb²⁺ and Ca²⁺: If the Ca²⁺-binding site is relatively rigid and crowded with protein ligands, then Pb²⁺, upon binding, preserves the native metal-binding site geometry and at low concentrations, can act as an activator of the host protein. If the Ca²⁺-binding site is flexible and consists of only a few protein ligands, then Pb²⁺ can displace Ca²⁺ and deform the native metal-binding site geometry, resulting in protein malfunction.