In the absence of donor tissues, conduits are needed for axons to regenerate across nerve defects, yet single-channel conduits may result in axonal dispersion, and multichannel synthetic polymer conduits have failed due to dimensional instability. The goal of this study was to create a robust collagen-based nerve conduit with multiple submillimeter-diameter channels to facilitate nerve guidance. Toward this goal, we have developed a novel multistep molding technique to create single-, four-, and seven-channel conduits from collagen and examined the effects of crosslinking with 0-60mM (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC] in N-hydroxysuccinimide) on geometric, enzymatic, and thermal stability, mechanical properties, and cellular behavior. Multichannel collagen conduits crosslinked with 30mM EDC and 10mM N-hydroxysuccinimide demonstrated low degradation rate (similar to 10% at 2 days), high shrinkage temperature (>75 degrees C), and constant channel morphology out to 30 days in saline. Neurite outgrowth remained unaffected from cultured dorsal root ganglia explants seeded on collagen scaffolds with up to 30mM EDC crosslinking. Compared with single-channel conduits, multichannel collagen conduits showed superior structural compressive, tensile, and bending stiffness. Taken together, these results suggest that the crosslinked multichannel collagen conduits possess favorable material and mechanical properties for nerve regeneration applications.