There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton which may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, hearts of CD73 knockout mice (deficient in extracellular adenosine production) contained elevated levels of detyrosinated microtubules compared to wild-type mice (195%; p<.05). In response to aortic banding, microtubules increased in wild-type hearts; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold stable triton insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic banded mice reduced hypertrophy and improved cardiac function compared to saline treated controls. These results indicate that microtubules contribute to cardiac dysfunction, and identifies for the first time a role for adenosine in regulating cardiomyocyte microtubule dynamics.