Cardiac myocytes from the mdx mouse, the mouse model of Duchenne muscular dystrophy, exhibit t-tubule disarray and increased calcium sparks, but a unifying molecular mechanism has not been elucidated. Recently, improper trafficking of junctophilin (JPH)-2 on an altered microtubule network caused t-tubule derangements and calcium mishandling in a pressure-overload heart failure model. Mdx cardiac myocytes have microtubule abnormalities, but how this may affect JPH-2, t-tubules, and calcium handling has not been established. Here, we investigated the hypothesis that an inverse relationship between microtubules and JPH-2 underlies t-tubule disruptions and calcium mishandling in mdx cardiac myocytes. Confocal microscopy revealed t-tubule disorganization in mdx cardiac myocytes. Quantitative Western blot analysis demonstrated JPH-2 was decreased by 75% and showed an inverse hyperbolic relationship with α- and β-tubulin, the individual components of microtubules, in mdx hearts. Colchicine-induced microtubule depolymerization normalized JPH-2 protein levels and localization, corrected t-tubule architecture, and reduced calcium sparks. In summary, these results suggest microtubule-mediated misregulation of JPH-2 causes t-tubule derangements and altered calcium handling in mdx cardiac myocytes.