van der Waals (vdW) epitaxy can be used to grow epilayers with different symmetries on graphene, thereby imparting unprecedented properties in graphene owing to formation of anisotropic superlattices and strong interlayer interactions. Here, we report in-plane anisotropy in graphene by vdW epitaxially grown molybdenum trioxide layers with an elongated superlattice. The grown molybdenum trioxide layers led to high p-doping of the underlying graphene up top= 1.94 × 1013cm−2regardless of the thickness of molybdenum trioxide, maintaining a high carrier mobility of 8155 cm2V−1s−1. Molybdenum trioxide–induced compressive strain in graphene increased up to −0.6% with increasing molybdenum trioxide thickness. The asymmetrical band distortion of molybdenum trioxide–deposited graphene at the Fermi level led to in-plane electrical anisotropy with a high conductance ratio of 1.43 owing to the strong interlayer interaction of molybdenum trioxide–graphene. Our study presents a symmetry engineering method to induce anisotropy in symmetric two-dimensional (2D) materials via the formation of asymmetric superlattices with epitaxially grown 2D layers.