Maternal periconceptional alcohol (PC-EtOH) exposure in the rat causes fetal growth restriction and sex-specific changes to placental morphology in late gestation. This may derive from perturbations to the pre-implantation embryo affecting its capacity to form a placenta. This study examined cell allocation in the pre-implantation embryo and trophectodermal (TE) derivatives after PC-EtOH exposure. Sprague Dawley dams were administered 12.5% v/v EtOH or a control liquid diet from 4 days prior (E-4) to 4 days after conception (E4). Female, but not male exposed PC-EtOH embryos showed reduced inner cell mass count (P<0.05), with no changes to total or TE cell count. TE nuclear CDX2 fluorescence was reduced in PC-EtOH embryos of both sexes (PTrt<0.0001), suggesting delayed formation of the TE. To assess trophoblast differentiation and migration of PC-EtOH embryos, a subset of in vivo derived embryos at E5 were cultured in vitro for 6 days. Cultures of PC-EtOH exposed E5 blastocysts showed decreased rate of trophoblast outgrowth (P<0.05), and number of trophoblasts (P<0.01) in both sexes. Investigation of parietal trophoblast giant cells (P-TGCs), the largest and most invasive trophoblasts, showed reduced P-TGC numbers in PC-EtOH females only (P<0.05), suggestive of perturbed differentiation. Analysis of DNA content showed no change with PC-EtOH exposure. PC-EtOH females had reduced expression of Prl4a1, a gene exclusively expressed by TGCs for communication with decidual natural killer cells, required for spiral artery remodelling in later placentation. Preliminary analysis of the depth of TGC invasion into the endometrium, suggests PC-EtOH may have also impaired invasion in vivo. In summary, PC-EtOH can alter pre-implantation development, trophoblast differentiation and invasive capacity, particularly in females, which is likely to contribute to placental defects, fetal growth restriction and programming of adult disease.