AbstractZinc‐mediated inhibition is implicated in global caspase regulation, with relief of zinc‐mediated inhibition central to both small‐molecule and natively induced caspase activation. As an initiator, caspase‐9 regulates the upstream stages of the apoptotic caspase cascade, making it a critical control point. Here we identify two distinct zinc‐binding sites on caspase‐9. The first site, composed of H237, C239, and C287, includes the active site dyad and is primarily responsible for zinc‐mediated inhibition. The second binding site at C272 is distal from the active site. Given the amino‐acid conservation in both regions, these sites appear to be present across the caspase family underscoring the importance of zinc‐mediated regulation of this class of enzymes.