OBJECTIVE: To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. DESIGN: Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. SETTING: Laboratory study. PATIENT(S): None. INTERVENTION(S): Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. MAIN OUTCOME MEASURE(S): Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. RESULT(S): Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. CONCLUSION(S): Liarozole decreased TGF-β3 and TGF-β3-mediated extracellular matrix expression in a 3D uterine leiomyoma culture system.