The treatment and repair of serious peripheral nerve injuries remain challenging in the clinical practice, while the application of multifunctional nerve guidance conduits (NGCs) based on naturally derived polymers has attracted much attention in recent years because of their excellent physicochemical properties and biological characteristics. Flammulina velutipes (Curt. ex FV) is a popular edible mushroom characterized by hollow tubular structures, antibacterial activities, and high nutritional properties. In this study, FV is utilized to construct NGCs (labeled FVC) via a freeze-drying technique without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC were assessed both in vitro and in vivo. FVC is composed of hollow tubes and evenly irregular interconnected micropores with 73.8 ± 5.5% porosity and 476.1 ± 12.9 μm hollow tube diameter. The inner surface of the FVC presents multiple microgrooves elongated parallel to the long axis. Moreover, FVC possessed strong antibacterial activity and could inhibit Gram-positive Staphylococcus aureus growth by up to 96.0% and Gram-negative Escherichia coli growth by up to 94.8% in vitro. FVC exhibited excellent biocompatibility and effectively promoted PC-12 cell proliferation and elongation in vitro. When applied to repair critical-sized sciatic nerve defects, FVC could effectively stimulate nerve functional recovery and axonal outgrowth in a rat model. Interestingly, Western blot analysis indicated that growth-associated protein 43 (GAP-43) had increased expression levels in the FVC group compared with the autograft group. This result suggested that by activating the Janus activated kinase2 (JAK2)/Phosphorylation ofsignal transducer and activator of transcription-3 (STAT3) signaling pathway, FVC upregulated Phosphorylation of signal transducer and activator of transcription-3 (P-STAT3) in vivo, resulting in the secretion of GAP-43. Collectively, a natural NGC FVC was fabricated based on FV without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC provide new insights for its further optimization and application in the field of nerve tissue engineering.