Relaxation abnormalities are prevalent in heart failure, and contribute to clinical outcomes. Disruption of Ca²⁺ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca²⁺ transients. We sought to speed cardiac relaxation by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca²⁺ buffer normally expressed in fast twitch skeletal muscle. A key feature of Parv’s function resides in its Ca²⁺/Mg²⁺ binding affinities that account for delayed Ca²⁺ buffering in response to the intracellular Ca²⁺ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca²⁺ content, without otherwise altering intracellular Ca²⁺ homeostasis. At high physiologic mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv’s delayed Ca²⁺ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca²⁺. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca²⁺ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationship of Parv in the heart, or engineering novel Parv-based Ca²⁺ buffers with modified Mg²⁺ and Ca²⁺ affinities to limit systolic Ca²⁺ buffering, may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.