The best understood “fight or flight” mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by β-adrenergic receptor (βAR) agonist stimulation. HCN4 conducts an inward “pacemaker” current (I_f) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I_f-independent pathways are critical to SAN fight or flight responses. The multifunctional Ca²⁺ and calmodulin-dependent protein kinase II (CaMKII) is a downstream signal in the βAR pathway that activates Ca²⁺ homeostatic proteins in ventricular myocardium. Mice with genetic, myocardial and SAN cell CaMKII inhibition have significantly slower HRs than controls during stress, leading us to hypothesize that CaMKII actions on SAN Ca²⁺ homeostasis are critical for βAR agonist responses in SAN. Here we show that CaMKII mediates ISO HR increases by targeting SAN cell Ca²⁺ homeostasis. CaMKII inhibition prevents ISO effects on SAN Ca²⁺ uptake and release from intracellular sarcoplasmic reticulum (SR) stores that are necessary for increasing DDR. CaMKII inhibition has no effect on the ISO response in SAN cells when SR Ca²⁺ release is disabled and CaMKII inhibition is only effective at slowing HRs during βAR stimulation. These studies show the tightly coupled, but previously unanticipated, relationship of CaMKII to the βAR pathway in fight or flight physiology and establish CaMKII as a critical signaling molecule for physiological HR responses to catecholamines.