The development of three-dimensional, spatially defined neuronal cultures that mimic chemical and physical attributes of native tissue is of considerable interest for various applications, including the development of tailored neuronal networks and clinical repair of damaged nerves. Here, the use of multiphoton excitation to photocrosslink protein microstructures within three-dimensional, optically transparent hydrogel materials, such as those based on hyaluronic acid, is reported. Multiphoton excitation confines photocrosslinking to a three-dimensional voxel with submicron spatial resolution, enabling fabrication of protein matrices with low- to sub-micrometer feature sizes by scanning the focus of a laser relative to the reagent solution. These methods can be used to create complex three-dimensional architectures that provide both chemical and topographical cues for cell culture and guidance, providing for the first time a means to direct cell adhesion and migration on size scales relevant to in vivo environments. Using this approach, guidance of both dorsal root ganglion cells (DRGs) and hippocampal neural progenitor cells (NPCs) along arbitrary, three-dimensional paths is demonstrated.