Pivotal to successful mammalian reproduction is the ability of a developing embryo to implant to the uterine wall and establish a nutrient supply via placentation. Herein, we have examined the potential epigenetic regulation of human trophoblastic cell migration and invasion by use of the choriocarcinoma cell line, BeWo. Treatment of BeWo cells with a DNA methyltransferase inhibitor, 5'-aza-2'-deoxycytidine (AZA), resulted in conversion of cell morphology to a nonmigratory phenotype. This was exemplified by the ability of AZA to prevent BeWo cell migration in wound healing and transwell migration assays. AZA consequently inhibited BeWo cell invasion through reconstituted basement membrane. Examination of components of the adherens junction complex pivotal for determination of cell phenotype revealed that AZA specifically increased the mRNA level of E-cadherin and plakoglobin (gamma-catenin), but not alpha-catenin and beta-catenin. AZA also increased the gene promoter activity of both plakoglobin and E-cadherin. Protein levels of both plakoglobin and E-cadherin were increased by AZA, and AZA enhanced their localization to sites of intercellular contact. Forced expression of plakoglobin and E-cadherin abrogated BeWo cell migration, indicative that repression of these genes was required for BeWo cell migration. Small interfering RNA-mediated depletion of the individual DNA methyltransferase (DNMT) molecules did not affect plakoglobin and E-cadherin promoter activity or BeWo cell migration. However, increases in plakoglobin and E-cadherin promoter activity and inhibition of BeWo cell migration was achieved with small interfering RNA-mediated depletion of both DNMT-3a and DNMT-3b. Epigenetic regulation of plakoglobin and E-cadherin is therefore pivotal for appropriate trophoblastic invasion in vitro.