Pin 1 is a peptidyl-prolyl isomerase inhibitor related to cyclophilin A and FK506 binding protein (FKBP). Juglone (5-hydroxy-1,4-naphthoquinone) is a natural inhibitor of Pin 1 with anti-inflammatory and antifibrotic properties. We evaluated the role of Pin 1 in renal fibrogenesis by evaluating the effects of juglone on epithelial to mesenchymal transition (EMT) and fibrogenesis in the rat unilateral ureteral obstruction (UUO) model and normal rat tubular epithelial cells (NRK52E). After 2 weeks of UUO, immunoblot analyses demonstrated that juglone (0.25 and 1 mg/kg/24 h) inhibited the deposition of matrix (alpha-smooth muscle actin (SMA), collagen type III and vimentin) and the activation of signaling pathways involved in fibrogenesis (phospho-smad2) and stress response (phospho-heat shock protein (HSP)27). Juglone also reduced EMT (alpha-SMA and E-cadherin dual staining) and oxidative stress (Mn superoxide dismutase (SOD) and NAPDH oxidase 2 (Nox-2) dual staining) in the obstructed kidney. There was no difference in Pin 1 levels between treatment and control groups. Pin 1 activity was significantly decreased in obstructed kidneys regardless of treatment status. In vitro, juglone (1 muM) significantly decreased alpha-SMA and p-smad levels compared to vehicle. Juglone attenuates fibrogenesis via Pin 1-independent mechanisms in the UUO model. The antifibrotic effects of juglone may result from the inhibition of smad2 and oxidative stress.