Contractile dysfunction in pressure overload-hypertrophied myocardium has been attributed in part to increased density of a stabilized cardiocyte microtubule network. The present study, the first to employ wild-type and mutant tubulin transgenes in a living animal, addresses this microtubule hypothesis directly by defining contractile mechanics of the normal and hypertrophied left ventricle (LV) and its constituent cardiocytes from transgenic mice having cardiac-restricted replacement of native 4-tubulin with 1-tubulin mutants that had been selected for their effects on microtubule stability and thus microtubule network density. In each case, replacement of cardiac 4-tubulin with mutant HA-tagged 1-tubulin was well tolerated in vivo. When LVs in intact mice and cardiocytes from these same LVs were examined in terms of contractile mechanics, baseline function was reduced in mice with genetically hyperstabilized microtubules, and hypertrophy-related contractile dysfunction was exacerbated. However, in mice with genetically hypostabilized cardiac microtubules, hypertrophy-related contractile dysfunction was ameliorated. Thus, in direct support of the microtubule hypothesis, we show here that cardiocyte microtubule network density, as an isolated variable, is inversely related to contractile function in vivo and in vitro, and microtubule instability rescues most of the contractile dysfunction seen in pressure overload-hypertrophied myocardium.