Stress-activated kinases p38 MAPK and JNK promote renal fibrosis; however, how the pathways by which these kinases are activated in kidney disease remain poorly defined. Apoptosis signal-regulating kinase 1 (ASK1/MAPKKK5) is a member of the MAPKKK family that can induce activation of p38 and JNK. The present study examined whether ASK1 induces p38/JNK activation and renal fibrosis in unilateral ureteric obstruction (UUO) using wild-type (WT) and Ask1-deficient (Ask1(-/-)) mice. Basal p38 and JNK activation in WT kidneys was increased three- to fivefold in day 7 UUO mice in association with renal fibrosis. In contrast, there was no increase in p38 activation in Ask1(-/-) UUO mice, whereas JNK activation was only partially increased. The progressive increase in kidney collagen (hydroxyproline) content seen on days 7 and 12 of UUO in WT mice was significantly reduced in Ask1(-/-) UUO mice in association with reduced alpha-smooth muscle actin-positive myofibroblast accumulation. However, cultured WT and Ask1(-/-) renal fibroblasts showed equivalent proliferation and matrix production, indicating that ASK1 acts indirectly on fibroblasts. Tubular epithelial cells are the main site of p38 activation in the obstructed kidney. Angiotensin II and H2O2, but not IL-1 or lipopolysaccharide, induced p38 activation and upregulation of transforming growth factor-beta1, platelet-derived growth factor-B, and monocyte chemoattractant protein-1 production was suppressed in Ask1(-/-) tubular epithelial cells. In addition, macrophage accumulation was significantly inhibited in Ask1(-/-) UUO mice. In conclusion, ASK1 is an important upstream activator of p38 and JNK signaling in the obstructed kidney, and ASK1 is a potential therapeutic target in renal fibrosis.