To achieve multifunctional materials using a single kind of ingredient or particle, it is desired to develop simple methods to prepare anisotropic nanoparticles, such as mushroom-like nanoparticles, which can be applied to many fields. Here, we investigate the wetting of polymer nanospheres on the open ends of the nanopores of anodic alumina oxide templates, which offer asymmetric wetting environments, to fabricate mushroom-like anisotropic polymer nanoparticles with controllable morphologies. These nanostructures have special dynamic behaviors in aqueous solutions. In addition, we prepare mushroom-like Janus nanoparticles by altering the fabrication processes. This work not only demonstrates a novel method to prepare anisotropic polymer nanoparticles but also provides a detailed understanding of the wetting behaviors under asymmetric environments.