The role of differentiated trophoblast glycogen cells (GCs) in the ectoplacental cone (EPC) has not been elucidated yet. Recently, GC progenitors have been shown to be present from embryonic day 7.5 (E7.5), but glycogen is found in GC only from E10.5. Herein, we investigated the origin, localization and characterization of mouse GCs in EPC and their relationship with blood cells and trophoblast giant cells (TGCs) during placentation. Implantation sites (E5.5-E12.5) were processed for histological studies, histochemical detection (glycogen) and immunohistochemical staining (Ki67). Three-dimensional reconstruction of the EPC was obtained from suitably oriented embryos at E7.5. Our findings evidence that GCs are present and assembled in clusters from E6.5 to E12.5, and that they exhibit the classic vacuolated appearance and contain PAS-positive glycogen, which is amylase-sensitive and acetylation-resistant. In fact, only GCs were stained after acetylation, confirming unequivocally their presence in tissues. At E6.5, GCs showed numerous mitoses and vacuoles with scattered glycogen particles. At E7.5, GCs showed low numbers of mitoses and abundant vacuoles full of glycogen. During E7.5-E8.5, GCs were in close proximity to TGCs, and cells were intercalated by thin maternal blood spaces; placental GCs lost maternal blood contact during E9.5-E12.5. Our results indicate that GCs are originated and proliferate in the upper portion in the midregion of EPC at E6.5, and that at E7.5-E8.5 they show consistent glycogen deposits, which are likely metabolized to glucose. This compound may be directly transferred to circulating maternal blood, and used as a source of energy by GCs and TGCs during placentation.