Post-translational detyrosination of microtubules (dTyr) promotes mechanical resistance to cardiac contraction, but the role of dTyr in heart failure has not previously been investigated. Here, Chen et al. analysed left ventricular tissue of failing and non-failing human hearts, which revealed that upregulation and stabilization of the cytoskeleton is a prominent feature of end-stage heart failure. Moreover, the microtubule network was highly proliferated and detyrosinated in failing versus non-failing myocytes, which increased myocyte stiffness and impeded contractility. Pharmacological suppression of dTyr-microtubules, or adenoviral overexpression of tubulin tyrosine ligase, significantly lowered stiffness and enhanced contractility in failing, human cardiomyocytes.