In this work, the influence of surface topography on protein crystallization over Nafion is investigated. Two types of Nafion-based membranes were modified by soft lithographic techniques in order to create different topographies at the micro and nano scale and subsequently tested. From an analysis of the induction time, nucleation, and crystal growth rate of trypsin from bovine pancreas, all of the patterned Nafion-based membranes show an enhanced nucleation and crystal growth. To provide additional insight into the experimental observations, the wettability properties of the prepared samples and the ratio of the Gibbs free energy of heterogeneous nucleation to homogeneous nucleation were evaluated. The crystallization outcome results from the combined effect of both the structural and chemical properties of the nucleant Nafion surface.