The heparan sulfate proteoglycan glypican-1 is essential as a co-receptor for heparin binding growth factors, such as HB-EGF and FGF-2, in pancreatic cancer cells. In the present study, the role of glypican-1 in the regulation of TGF-beta signaling was investigated. Colo-357 pancreatic cancer cells were stably transfected with a full-length glypican-1 antisense construct. Cell growth was determined by MTT and soft agar assays. TGF-beta1 induced p21 expression and Smad2 phosphorylation were analyzed by immunoblotting. PAI-1 promoter activity was determined by luciferase assays. Down-regulation of glypican-1 expression by stable transfection of a full-length glypican-1 antisense construct resulted in decreased anchorage-dependent and -independent cell growth in Colo-357 pancreatic cancer cells and attenuated TGF-beta1 induced cell growth inhibition, Smad2 phosphorylation, and PAI-1 promoter activity. There was, however, no significant difference in TGF-beta1 induced p21 expression and Smad2 nuclear translocation. In conclusion, glypican-1 is required for efficient TGF-beta1 signaling in pancreatic cancer cells.