The pathogenesis of ketamine-induced cystitis (KC) remains unclear. In this study, bladder microvascular injury was investigated as a possible contributing mechanism. A total of 36 KC patients with exposure to ketamine for more than 6 months, and 9 control subjects, were prospectively recruited. All participants completed questionnaires, including the O'Leary-Sant interstitial cystitis symptom index (ICSI) and the interstitial cystitis problem index (ICPI). All KC patients received a urodynamic study and radiological exams. Bladder tissues were obtained from cystoscopic biopsies in the control group and after hydrodistention in the KC group. Double-immunofluorescence staining of N-methyl-d-aspartate receptor subunit 1 (NMDAR1) and the endothelial marker, cluster of differentiation 31 (CD31), was performed to reveal the existence of NMDAR1 on the endothelium. Electron microscopy (EM) was applied to assess the microvascular change in the urinary bladder and to measure the thickening of the basement membrane (BM). A proximity ligation assay (PLA) was used to quantify the co-localization of the endothelial CD31 receptor and the mesenchymal marker [fibroblast-specific protein 1 (FSP-1)]. The Mann-Whitney U test and Spearman's correlation coefficient were used for statistical analysis. The mean ICSI [14.38 (± 4.16)] and ICPI [12.67 (± 3.54)] scores of the KC group were significantly higher than those (0 and 0, respectively) of the control group (both p < 0.001). The KC patients had decreasing cystometric bladder capacity (CBC) with a mean volume of 65.38 (± 48.67) mL. NMDAR1 was expressed on endothelial cells in both groups under immunofluorescence staining. Moreover, KC patients had significant BM duplication of microvessels in the mucosa of the urinary bladder under EM. The co-expression of the endothelial marker CD31 and mesenchymal marker FSP1 was significantly stained and calculated under PLA. In conclusion, microvascular injury and mesenchymal phenotypic alteration of endothelial cells can potentially contribute to KC-induced bladder dysfunction.