Nerve conduits (NCs) with multiple longitudinally aligned channels, being mimicking the natural nerves anatomical structure, have been attracted more and more attentions. However, some specific structural parameters of a conduit that would be beneficial for further improvement of neural tissue regeneration were not comprehensively considered. Using a systematized device and combining low-pressure injection molding and thermal-induced phase separation, we fabricated 33-channel NCs (outer diameter 3.5 mm, channel diameter 200 μm) with different well-defined microscopic features, including NCs with a nano-fibrous microstructure (NNC), NCs with microspherical pores and nano-fibrous pore walls (MNC), and NCs with a ladder-like microstructure (LNC). The porosities of these NCs were ∼90% and were independent of the fine microstructures, whereas the pore size distributions were clearly distinct. The adsorption of bovine serum albumin for the NNC was a result of having the highest specific surface area, which was 3.5 times that of the LNC. But the mechanical strength of NNC was lower than that of two groups because of a relative high crystallinity and brittle characteristics. In vitro nerve stem cells (NSCs) incubation revealed that 14 days after seeding the NSCs, 31.32% cells were Map2 positive in the NNC group, as opposed to 15.76% in the LNC group and 23.29% in the MNC group. Addition of NGF into the culture medium, being distinctive specific surface area and a high adsorption of proteon for NNC, 81.11% of neurons derived from the differentiation of the seeded NSCs was obtained. As a result of imitating the physical structure of the basement membrane of the neural matrix, the nanofibrous structure of the NCs has facilitated the differentiation of NSCs into neurons.