Significance Our in vitro analyses reveal that dystrophin, the protein absent in Duchenne muscular dystrophy patients, binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin, the autosomal homologue of dystrophin thought to mirror many known functions of dystrophin, has no activity in either assay. We also report that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, physical inactivity after mild exercise, or loss of torque production after in vivo eccentric contraction in dystrophin-deficient skeletal muscle. Finally, our data demonstrate that microtubule lattice disorganization contributes to the greater eccentric contraction-induced injury experienced by dystrophin-deficient skeletal muscle, demonstrating a phenotype of dystrophin deficiency that utrophin-based therapy may not be able to correct.