Purpose: To investigate the anisotropic elasticity of soft tissues using MR elastography (MRE) combined with diffusion tensor imaging (DTI). Materials and Methods: The storage moduli parallel (μ∥) and perpendicular (μ⊥) to the local fiber orientation were calculated assuming a transversely isotropic model. The local fiber orientation was provided by DTI. The proposed technique was validated against rheometry using anisotropic viscoelastic phantoms with various fiber volume fractions (Vf = 0%, 15%, and 35%) and bovine skeletal muscle samples. Results: The anisotropic ratio (μ∥/μ⊥) as measured by MRE correlated well with rheometry for all samples (R2 = 0.809). The combined MRE/DTI technique was also able to differentiate different levels of mechanical anisotropy with the mechanical anisotropy (μ∥/ μ⊥) of the Vf = 35% phantoms being significantly higher than the Vf = 15% and the isotropic (Vf = 0%) phantoms. The bovine muscle samples showed significantly higher mechanical anisotropy than all phantoms. Conclusion: This study has demonstrated the feasibility of the proposed imaging technique for characterizing mechanical anisotropy of anisotropic materials and biological tissues, and validated the mechanical anisotropy results. J. Magn. Reson. Imaging 2013;37:217-226. © 2012 Wiley Periodicals, Inc.