Extravillous trophoblast (EVT) cells of the human placenta progressively lose their proliferative activity in situ as EVT cell columns migrate into and invade the decidua. It remains unclear whether this is due to a terminal differentiation of EVT cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation by decidua-derived factors, or both mechanisms. Our earlier studies provided evidence for a negative regulation by a decidua-derived factor, transforming growth factor (TGF)-β, which inhibited proliferation, migration, and invasiveness of first-trimester EVT cells in vitro. We further discovered that decidua also produces decorin, a proteoglycan that binds TGF-β (and in some cases, inactivates TGF-β), which is colocalized with TGF-β in the decidual extracellular matrix. The present study used in vitro-propagated EVT cell lines to examine whether EVT cells retain their capacity for proliferation after the process of invasion; and whether decorin exerts any effect on EVT cell proliferation, migration, or invasiveness in a TGF-β-dependent or TGF-β-independent manner. We also examined whether trophoblastic cancer (choriocarcinoma) JAR and JEG-3 cells responded to decorin in a similar manner. Proliferation was measured using a colorimetric (MTT) cellularity assay and immunolabeling for the Ki-67 proliferation marker. Migration and invasiveness were measured in transwells by the ability of cells to cross 8-μm pores of polycarbonate membranes in the absence or presence of an additional matrigel barrier. These experiments revealed three points. First, EVT cells retained limited but significant proliferative ability in vitro after invading matrigel. Second, that decorin alone blocked EVT cell proliferation in a dose-dependent manner. This effect remained unaffected in an additional presence of TGF-β, which exerted antiproliferative effects on its own. The antiproliferative effect of decorin was explained by an up-regulation of the p21 protein. Third, that decorin alone or TGF-β alone exerted antimigratory and anti-invasive effects on EVT cells, but the addition of TGF-β to decorin did not alter decorin action. And fourth, that choriocarcinoma cells were resistant to antiproliferative, antimigratory, and anti-invasive effects of decorin. These results suggest 1) that the invasive function of EVT cells is not associated with a terminal differentiation into a noncycling state; 2) that proliferation, migration, and invasiveness of EVT cells within the decidua are independently controlled by two decidual products, TGF-β and decorin (decorin in the decidual extracellular matrix may serve as a storage mechanism for TGF-β in an inactive state and may be activated by EVT cell proteolytic mechanisms, thus preventing overinvasion); and 3) that choriocarcinoma cells are refractory to negative regulation by both decidua-derived factors.