Aspartyl-asparaginyl-β-hydroxylase (AAH) regulates cell motility and invasiveness by enhancing Notch signaling. Invasive trophoblastic cells, which mediate placentation, normally express high levels of AAH. Previously, we showed that ethanol-impaired placentation is associated with reduced AAH expression. The present study determines the degree to which inhibition of AAH expression is sufficient to impair functions required for placentation. Immortalized, first trimester-derived, human trophoblastic cells (HTR-8/SVneo) were transfected with siRNA targeting AAH (siRNA-AAH) or no specific sequences (siRNA-Scr) using the Amaxa electroporation system. Directional motility was measured using an ATP luminescence-based assay. For in vivo studies, we microinjected siRNA-AAH or siRNA-Scr directly into the implantation sites (mesometrial triangle) of gestation-day-17, Long Evans pregnant rats, and harvested placentas 24 h later for histologic and molecular studies. siRNA-AAH transfection reduced AAH expression and directional motility in HTR-8/SVneo cells. In vivo delivery of siRNA-AAH reduced AAH expression and mean number of invasive trophoblastic cells at the implantation site. These adverse effects of siRNA-AAH were associated with impaired fetal growth and significantly reduced expression of Notch-signaling network genes. AAH is an important, positive regulator of trophoblastic cell motility, and inhibition of AAH in vivo leads to impaired implantation and fetal growth, and alters Notch-signaling mechanisms, similar to the effects of chronic ethanol exposure.