<p>Chronic fibrosis replaces functional organ tissue with scar tissue by overproduction of a thick and stiff extracellular matrix. Bladder fibrosis decreases bladder compliance, ultimately resulting in overactive bladder. The phenoconversion of fibroblasts into myofibroblasts is the defining feature of fibrosis. Recently, regionally distinct populations of bladder platelet-derived growth factor receptor alpha positive (PDGFRα⁺) cells were identified as fibroblasts. Because of this heterogeneity, the identity of the bladder fibroblast cells that undergo phenotypic conversion into myofibroblasts is not clear. The current study utilized cyclophosphamide (CYP)-induced bladder inflammation to identify and characterize bladder PDGFRα⁺ cells that become myofibroblasts. We found that suburothelial PDGFRα⁺ cells and detrusor PDGFRα⁺ cells display different gene expression profiles. Suburothelial PDGFRα⁺ cells are more abundant than detrusor PDGFRα⁺ cells and express higher levels of fibrosis-related genes. CYP-treatment increased the number of suburothelial PDGFRα⁺ cells, increased <i>Pdgfra, Col1a1, and Fn1</i> transcription in suburothelial PDGFRα⁺ cells, and increased α-smooth muscle actin, collagen, and fibronectin protein expression. CYP-treatment likely activated TNF-α and TGF-ß pathways, as indicated by nuclear translocation of SMAD2, SMAD3, and NFκB. In conclusion, we identify suburothelial PDGFRα^<b>+</b> cells as the fibroblast population which convert into myofibroblasts via activation of TNF-α and TGF-ß signaling pathways, due to bladder inflammation.</p>