Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.