Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47^phox in young (5-6 mo) and aged (26-28 mo) FVB wild-type (WT) and cardiac-specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR₉₀, reduced tolerance to high stimulus frequency, and slowed intracellular Ca²⁺ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47^phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging-induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol-induced superoxide generation and apoptosis. In addition, aging-associated prolongation in TR₉₀ was blunted by the Rho kinase inhibitor Y-27632. Collectively, our data demonstrated that metallothionein may alleviate aging-induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice.--Yang, X., Doser, T. A., Fang, C. X., Nunn, J. M., Janardhanan, R., Zhu, M., Sreejayan, N., Quinn, M. T., Ren, J. Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress