Background Sodium-calcium (Ca²⁺) exchanger isoform 1 (NCX1) is the dominant Ca²⁺ efflux mechanism in cardiomyocytes and is critical to maintaining Ca²⁺ homeostasis during excitation-contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen-inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation-contraction coupling and intracellular Ca²⁺ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury. Methods and Results We used the α-myosin heavy chain promoter (Myh6)-MerCreMer promoter to create a tamoxifen-inducible cardiac-specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca²⁺ increased despite adaptive reductions in Ca²⁺ current and action potential duration and compensatory increases in excitation-contraction coupling gain, sarcoplasmic reticulum Ca²⁺ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca²⁺ normalized and SR Ca²⁺ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular-related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion. Conclusions Tamoxifen-inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca²⁺ influx, increasing alternative Ca²⁺ efflux pathways, and increasing excitation-contraction coupling gain to maintain contractility at the cost of mild Ca²⁺-activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.