Antithrombin, a key serpin family regulator of blood coagulation proteases, is transformed into a potent antiangiogenic factor by limited proteolysis or mild heating. Here, we show by cDNA microarray, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting analyses that the expression of the proangiogenic heparan sulfate proteoglycan (HSPG), perlecan, but not other HSPGs, is dramatically down-regulated in human umbilical vein endothelial cells (HUVECs) treated with antiangiogenic cleaved and latent forms of antithrombin but not with the native form. Down-regulation of perlecan expression by cleaved and latent antithrombins was observed in both basic fibroblast growth factor (bFGF)-stimulated and unstimulated cells, whereas the antiangiogenic antithrombins inhibited the proliferation of only bFGF-stimulated HUVECs by arresting cells at the G(1) cell cycle phase. The importance of perlecan expression levels in mediating the antiproliferative effect of the antiangiogenic antithrombins was suggested by the finding that transforming growth factor-beta 1, a potent stimulator of perlecan expression in endothelial cells, blocked the down-regulation of perlecan expression and antiproliferative activity of cleaved antithrombin on endothelial cells. The previously established key role of perlecan in mediating bFGF stimulation of endothelial cell proliferation and angiogenesis suggests that a primary mechanism by which antiangiogenic antithrombins exert their effects is through the down-regulation of perlecan expression.