The pathogenesis of detrusor underactivity (DU) is unclear, and the available therapeutic effects are unsatisfactory. We propose to find key molecules and pathways related to DU based on transcriptome sequencing. A rat model of bilateral pelvic nerve injury (BPNI) was established. Bladder tissues from the sham-operated group, 3 and 28 days after BPNI mapping, were taken for urodynamics, histopathology, and RNA-seq. An enrichment analysis of the screened differential expression genes was performed. Three days after BPNI, the results showed urodynamic features of overflow incontinence, while there was a recovery at 28 days after the operation. Masson staining revealed collagen deposition accompanied by progressive thickening of the smooth muscle layer as DU progressed. RNA-seq results suggested that a total of 1808 differentially expressed genes (DEGs) differed among the groups. RNA-seq and subsequent analysis confirmed that the cell cycle and immune response were significantly activated 3 days after BPNI, while extracellular matrix remodeling occurred 28 days after BPNI. Partial DEGs and pathways were verified by qRT-PCR. Validation of key proteins involved in cell cycle, inflammation, and fibrosis was performed by immunohistochemical staining and western blot, respectively. These molecular expression patterns at different time points after BPNI injury provide valuable insights into the search for therapeutic targets for DU.