β₁-adrenergic receptor (β₁AR) stimulation activates the classic cAMP/protein kinase A (PKA) pathway to regulate vital cellular processes from the change of gene expression to the control of metabolism, muscle contraction, and cell apoptosis. Here we show that sustained β₁AR stimulation promotes cardiac myocyte apoptosis by activation of Ca²⁺/calmodulin kinase II (CaMKII), independently of PKA signaling. β₁AR-induced apoptosis is resistant to inhibition of PKA by a specific peptide inhibitor, PKI14-22, or an inactive cAMP analogue, Rp-8-CPT-cAMPS. In contrast, the β₁AR proapoptotic effect is associated with non–PKA-dependent increases in intracellular Ca²⁺ and CaMKII activity. Blocking the L-type Ca²⁺ channel, buffering intracellular Ca²⁺, or inhibiting CaMKII activity fully protects cardiac myocytes against β₁AR-induced apoptosis, and overexpressing a cardiac CaMKII isoform, CaMKII-δC, markedly exaggerates the β₁AR apoptotic effect. These findings indicate that CaMKII constitutes a novel PKA-independent linkage of β₁AR stimulation to cardiomyocyte apoptosis that has been implicated in the overall process of chronic heart failure.