Aims To investigate the effects of injecting RNA interference (RNAi) lentiviruses targeting the muscarinic 3 (M-3) receptor gene into the bladder wall on bladder activity in rats with spinal cord injury (SCI). Methods Four M-3 RNAi lentiviruses were constructed and used to infect primary cultured bladder smooth muscle cells (BSMCs). Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to determine the optimal RNAi lentivirus with the highest interference efficiency. Female Wistar rats were subjected spinal cord transection at T9-10 and randomly divided into three groups (n = 8), namely, blank control, negative control, and experimental groups, and injected into the bladder wall with saline, negative control shRNA, and M-3 RNAi lentiviruses, respectively, 1 week after spinal cord transection. The normal rats were used as normal control group. Urodynamic parameters and bladder tissues were evaluated in the different groups. Results An M-3 RNAi lentivirus with the highest interference efficiency (78.9%) was constructed and identified. Three weeks after injecting M-3 RNAi lentiviruses into the bladder wall, Western blotting and qRT-PCR showed that the M-3 receptor was significantly downregulated in the experimental group. Cystometric evaluation suggested that downregulating M-3 receptor expression could substantially decrease basal pressure, residual volume, and non-voiding contraction number, increase intercontraction interval, and significantly improve bladder compliance in rats with SCI. Conclusion Injecting RNAi lentiviruses targeting the M-3 receptor gene into the bladder wall could effectively inhibit neurogenic detrusor overactivity (NDO) due to SCI. Thus, this approach may be a potential treatment for NDO in SCI.