The function of hypoxia-inducible factor-1alpha (HIF-1alpha) in chronic kidney disease is disputed. Here we report that interactions of HIF-1alpha with transforming growth factor-beta (TGF-beta) signaling may promote its fibrotic effects. Knockout of HIF-1alpha is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model. Transcriptional analysis of cultured renal cells showed that alpha2(I) collagen expression is directly regulated by HIF-1alpha binding to a functional hypoxia-responsive element in its promoter at -335 relative to the transcription start site. Activation of COL1A2 transcription by HIF-1alpha occurred in the absence of hypoxia and is strongly enhanced by TGF-beta signaling. TGF-beta, in addition to increasing HIF-1alpha levels, increased both HIF-1alpha binding to the COL1A2 promoter and HIF-1alpha N-terminal transactivation domain activity. These effects of TGF-beta on HIF-1alpha were inhibited in Smad3-null mouse embryonic fibroblasts, suggesting a requirement for Smad3. Phosphorylated Smad3 also associated with the -335 hypoxia-responsive element of the COL1A2 promoter independent of a Smad DNA binding sequence. Smad3 binding to the -335 hypoxia-responsive element required HIF-1alpha both in vitro and in kidney lysate from the disease model, suggesting formation of an HIF-1alpha-Smad3 transcriptional complex. Thus, HIF-1alpha-Smad3 has a novel interaction in glomerulosclerosis.