Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widely distributed endogenous neuropeptide, also occurring in the cardiovascular system. Among others, PACAP has been suggested as a cardioprotective factor. It has been shown that PACAP inhibits cardiac fibrosis and protects cardiomyocytes against oxidative stress and in vitro ischemia/reperfusion. The aim of the present study was to investigate whether PACAP is protective in doxorubicin-induced cell death of cardiomyocytes. Cardiomyocytes were exposed to 1 µM doxorubicin for 24 h, which resulted in a marked reduction of cell viability and a parallel increase of apoptotic cells assessed by MTT test and annexin V/propidium iodide flow cytometry assay. Co-incubation with 20 nM PACAP increased cell viability and reduced the percentage of apoptotic cells. Furthermore, doxorubicin increased the activation of caspase-3 and decreased the phosphorylation of Bad, while simultaneous PACAP treatment reduced the caspase-3 activation and increased the level of phospho-Bad. In summary, our present results demonstrate that PACAP effectively protects cardiomyocytes against doxorubicin-induced apoptotic cell death.