PurposeDiffusion magnetic resonance imaging (MRI) is increasingly used to characterize cardiac tissue microstructure, necessitating the use of physiologically relevant phantoms for methods development. Existing phantoms are generally simplistic and mostly simulate diffusion in the brain. Thus, there is a need for phantoms mimicking diffusion in cardiac tissue.Materials and MethodsA biomimetic phantom composed of hollow microfibers generated using co‐electrospinning was developed to mimic myocardial diffusion properties and fiber and sheet orientations. Diffusion tensor imaging was carried out at monthly intervals over 4 months at 9.4T. 3D fiber tracking was performed using the phantom and compared with fiber tracking in an ex vivo rat heart.ResultsThe mean apparent diffusion coefficient and fractional anisotropy of the phantom remained stable over the 4‐month period, with mean values of 7.53 ± 0.16 × 10‐4 mm2/s and 0.388 ± 0.007, respectively. Fiber tracking of the 1st and 3rd eigenvectors generated analogous results to the fiber and sheet‐normal direction respectively, found in the left ventricular myocardium.ConclusionA biomimetic phantom simulating diffusion in the heart was designed and built. This could aid development and validation of novel diffusion MRI methods for investigating cardiac microstructure, decrease the number of animals and patients needed for methods development, and improve quality control in longitudinal and multicenter cardiac diffusion MRI studies. J. MAGN. RESON. IMAGING 2016;43:594–600.