Skeletal muscle contains a highly hierarchical structure, leading to anisotropic mechanical properties, with varying morphological responses to mechanical loadings from different directions. However, this feature is rarely studied in clinical studies, mainly due to the challenges in quantifying muscle anisotropic mechanical properties in vivo. The aim of the current study was to quantify the anisotropic mechanical properties of skeletal muscle using an integrated approach combining multi-frequency magnetic resonance elastography (MRE) and diffusion tensor imaging (DTI). Muscle fascicle orientation was determined through DTI tractography. Direct inversion of the curl-based wave equation was used to quantify three complex-valued moduli (μ^*_⊥, μ^*_‖, and E^*_‖) assuming muscle as an incompressible transversely isotropic material. This approach was evaluated on one ex vivo muscle sample by comparing MRE-derived moduli to rheometry measurements, and further assessed in vivo in the ankle plantarflexors of nine able-bodied subjects. Consistency in the anisotropic ratio was observed between rheometry and MRE measurements in the ex vivo muscle sample, though discrepancies were noted in absolute shear moduli values. In vivo, the anisotropy of skeletal muscle was observed by the relationship of μ^*_⊥ ≠ 1/3E^*_‖ and μ^*_‖ ≠ 1/3E^*_‖ at different MRE driving frequencies with higher parallel shear modulus () than the perpendicular shear modulus (μ^*_⊥). This study demonstrated a promising approach for quantifying the muscle anisotropic mechanical properties in vivo, which can be useful in various clinical applications.