Background: An increasing amount of evidence has indicated that microRNAs (miRs) are involved in most biological conditions, including the neurogenic bladder (NB). However, to our knowledge, no studies have investigated these miR expressions in spinal cord-injured (SCI) rat NB. The goal of the study was to explore the miR expression profile in the SCI rat NB by next-generation sequencing (NGS). Methods: Female Wistar rats underwent spinal cord transection at T9-10 and were randomly divided into the SCI-1, SCI-2 and SCI-3 groups (n=5 for each group) whose bladder tissues were collected 1, 2, and 4 weeks after transection, respectively. The normal rats were used as the normal control (NC) group. MiRs microarray assays were used to detect the differentially expressed miRs between the groups by NGS, which was then verified by quantitative real-time polymerase chain reaction (qRT-PCR). Those significantly differently expressed miRs were analyzed with Gene Ontology categories and Kyoto Encyclopedia of Genes and Genomes bioinformatical analyses. Results: Compared with the NC group, 96, 28 and 51 miRs were downregulated in the rats' bladder in the SCI-1, SCI-2, and SCI-3 groups, respectively, and 133, 49, and 76 miRs were upregulated respectively. Specifically, miR-21-5p was the most significantly upregulated miR in all SCI groups. Also, 121 miRs (SCI-1 vs. SCI-2), 98 miRs (SCI-1 vs. SCI-3), and 26 miRs (SCI-2 vs. SCI-3) were of significantly different expression. Furthermore, a large set of genes implicated in essential signaling pathways were targeted by these miRs, including PI3K-Akt, MAPK, Rap1, and cGMP-PKG signaling pathways, along with the tight junction and metabolic pathways. Conclusions: This is the first demonstration of differentially expressed miRs, which may potentially serve as new molecular targets in the SCI rat NB.