Adipocyte differentiation is regulated largely through the actions of the PPAR nuclear receptor and the insulin signaling pathway. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPAR signaling pathways, and their association with adipocyte differentiation. Co-expression of PDK1 and PPAR1 in 293T cells stimulated PPAR response element (PPRE)-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPAR1 activity was comparable in magnitude to the coactivator AIB1, and was blocked by either the corepressor SMRT or dominant-negative PAX8-PPAR1. Heterologous Gal4-PPAR1 assays indicated that PDK1 interacted with the ligand-binding domain, and physically associated with PPAR1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPAR1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the -helical hydrophobic motifs in PDK1, L²⁶⁸XII and V³¹³XXLL, and expression of the -helical region encompassing these motifs stimulated PPRE-dependent transcription. PDK1-PPAR interaction was confirmed by chromatin immunoprecipitation (ChIP) analysis of the lipoprotein lipase (LPL) and adipocyte fatty acid-binding protein (aP2) promoters. In cells expressing PDK1 and PPAR, binding to PPAR response elements occurred, which was enhanced by treatment with a PPAR agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPAR agonist that was comparable to the response of PPAR1-transfected cells in the presence of agonist; expression of PDK1 and PPAR resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPAR agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPAR1 coactivator independently of its catalytic activity, and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.