The interruption of the β-adrenergic receptor signaling at the level of adenylyl cyclase (AC) by specifically knocking out (KO) the AC5 gene activates the RAF/MEK/ extracellular signal-regulated kinase (ERK) signaling pathway, delays bone and heart aging, and increases mean and maximum longevity in mice. However, the mechanisms involved in life extension in this animal model with increased longevity have not been clarified, although a decrease in oxidative stress has been proposed as mediator. Two traits link longevity and oxidative stress. Long-lived mammals and birds have a low rate of mitochondrial reactive oxygen species (mitROS) generation and a low degree of membrane fatty acid unsaturation, but these key factors have not been studied in AC5 KO mice. In the present investigation, male C57BL/6 mice were treated with the β-blocker atenolol in drinking water, and oxidative stress-related parameters were measured in the heart. Atenolol treatment did not change the rate of mitROS production and oxidative damage to mitDNA (8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG]), but strongly decreased the degree of fatty acid unsaturation and the peroxidizability index, mainly due to decreases in 22:6n-3 and 20:4n-6 and to increases in 18:1n-9, 16:1n-7 and 16:0 in the atenolol group. Protein oxidation and lipoxidation were lower in the atenolol group than in the controls. The mitochondrial complex I and IV content and the amount of p-ERK1/2 signaling proteins were significantly higher in the atenolol-treated than in the control animals. These results support the idea that the increased longevity of the AC5 KO mice can be due in part to an ERK signaling-mediated stress-resistance due to a decrease in fatty acid unsaturation, leading to lower lipid peroxidation and decreased lipoxidation-derived damage to cellular proteins.