This report describes a new approach for fabricating microchannels within three-dimensional electrospun constructs. These key features serve to mimic the fascicular architecture and fibrous extracellular matrix found in native nerve. Both electrospun fibers and multi-channeled structure nerve guides have become areas of increasing interest for their beneficial roles in nerve repair. However, to the best of our knowledge, this is the first report of a guide that incorporates both. Multiple parallel channels provide a greater number of defined paths and increased surface area compared to cylindrical guides. Additionally, the fibrous nature of electrospun fibers permits better mass transport than solid-walled constructs. The flexible fabrication scheme allows tailoring of nerve guide parameters such as channel diameters ranging from 33 to 176?mu m and various wall thicknesses. Channel and fiber structures were assessed by optical and electron microscope images. Geometric calculations estimated a porosity of over 85% for these guides with 16% or less from the channels. In vitro culture with Schwann cells demonstrated cellular infiltration into channels with restricted migration between fibers. Finally, cell proliferation and survival throughout the guide indicates that this design warrants future in vivo examination. Biotechnol. Bioeng. 2012; 109:15711582. (c) 2011 Wiley Periodicals, Inc.