Numerous studies have indicated that microgravity induces various changes in the cellular functions of neural stem cells (NSCs), and the use of microgravity to culture tissue engineered seed cells for the treatment of nervous system diseases has drawn increasing attention. The goal of this study was to verify the efficacy of collagen sponge-based 3-dimensional (3D) NSCs cultured in a rotary cell culture system (RCCS) in treating spinal cord injury (SCI). The Basso–Beattie–Bresnahan score, inclined plane test, and electrophysiology results all indicated that 3D cultured NSCs cultured in a RCCS had better therapeutic effects than those cultured in a traditional cell culture environment, suggesting that the microgravity provided by the RCCS could enhance the therapeutic effect of 3D cultured NSCs. Our study indicates the feasibility of combining the RCCS with collagen sponge-based 3D cell culture for producing tissue engineered seed cells for the treatment of SCI. This novel and effective method shows promise for application in cell-based therapy for SCI in the future.