Diabetic cystopathy (DCP) is a prevalent urinary complication in diabetes. Late-stage DCP can cause chronic urinary retention, significantly impacting the patient’s quality of life and even posing a risk to their lives. Therefore, it is crucial to investigate the pathogenesis and pathological characteristics of DCP. This study aims to utilize proteomics-related methods to uncover the pathogenesis of DCP by analyzing the differential protein expression profiles in the bladder tissue of diabetic rats and normal rats. Morphological analysis revealed notable changes in the bladder tissue of diabetic rats, including significant inflammatory cell infiltration and collagen fiber deposition. Immunohistological examination demonstrated increased apoptosis and decreased proliferation in the bladder tissue of diabetic rats. Quantitative proteomic analysis identified a total of 280 differentially expressed proteins between the two groups, with 193 proteins being down-regulated and 87 proteins being up-regulated. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated significant changes in protein expression profiles and signaling pathways in the bladder of diabetic rats. Additionally, transcription factor analysis revealed that the insulin-like growth factor-related protein family was the most affected. This study provides valuable evidence for identifying potential genes for the treatment of diabetic bladder.