Objectives We investigated the effects of erbB2 inhibition by anti-erbB2 antibody on cardiomyocyte survival and mitochondrial function. Background ErbB2 is an important signal integrator for the epidermal growth factor family of receptor tyrosine kinases. Herceptin, an inhibitory antibody to the erbB2 receptor, is a potent chemotherapeutic but causes cardiac toxicity. Methods Primary cultures of neonatal rat ventricular myocytes were exposed to anti-erbB2 antibody (Ab) (7.5 μg/ml) for up to 24 h. Cell viability, mitochondrial function, and apoptosis were measured using multiple complementary techniques. Results ErbB2 inhibition was associated with a dramatic increase in expression of the pro-apoptotic Bcl-2 family protein Bcl-xS and decreased levels of anti-apoptotic Bcl-xL. There was a time-dependent increase in mitochondrial translocation and oligomerization of bcl-associated protein (BAX), as indicated by 1,6-bismaleimidohexane crosslinking. The BAX oligomerization was associated with cytochrome c release and caspase activation. These alterations induced mitochondrial dysfunction, a loss of mitochondrial membrane potential (ψ) (76.9 ± 2.4 vs. 51.7 ± 0.1; p < 0.05; n = 4), a 35% decline in adenosine triphosphate levels (p < 0.05), and a loss of redox capacity (0.72 ± 0.04 vs. 0.64 ± 0.02; p< 0.01). Restoration of Bcl-xL levels through transactivating regulatory protein-mediated protein transduction prevented the decline in ψ mitochondrial reductase activity and cytosolic adenosine triphosphate. Conclusions Anti-erbB2 activates the mitochondrial apoptosis pathway through a previously undescribed modulation of Bcl-xL and -xS, causing impairment of mitochondrial function and integrity and disruption of cellular energetics.