Nerve conduits enhance nerve regeneration in the repair of long-distance peripheral nerve defects. To help optimize the effectiveness of nerve conduits for nerve repair, we developed a multi-step electrospinning process for constructing nerve guide conduits with aligned nanofibers. The alignment of the nerve guide conduits was characterized by scanning electron microscopy and fast Fourier transform. The mechanical performance of the nerve guide conduits was assessed by testing for tensile strength and compression resistance. The biological performance of the aligned fibers was examined using Schwann cells, PC12 cells and dorsal root ganglia in vitro. Immunohistochemistry was performed for the Schwann cell marker S100 and for the neurofilament protein NF200 in PC12 cells and dorsal root ganglia. In the in vivo experiment, a 1.5-cm defect model of the right sciatic nerve in adult female Sprague-Dawley rats was produced and bridged with an aligned nerve guide conduit. Hematoxylin-eosin staining and immunohistochemistry were used to observe the expression of ATF3 and cleaved caspase-3 in the regenerating matrix. The recovery of motor function was evaluated using the static sciatic nerve index. The number of myelinated fibers, axon diameter, fiber diameter, and myelin thickness in the distal nerve were observed by electron microscopy. Gastrocnemius muscle mass ratio was also determined. The analyses revealed that aligned nanofiber nerve guide conduits have good mechanical properties and can induce Schwann cells, PC12 cells and dorsal root ganglia to aggregate along the length of the nanofibers, and promote the growth of longer axons in the latter two (neuronal) cell types. The aligned fiber nerve conduits increased the expression of ATF3 and cleaved caspase-3 at the middle of the regenerative matrix and at the distal nerve segment, improved sciatic nerve function, increased muscle mass of the gastrocnemius muscle, and enhanced recovery of distal nerve ultrastructure. Collectively, the results show that highly aligned nanofibers improve the performance of the nerve conduit bridge, and enhance its effectiveness in repairing peripheral nerve defects.