Endoplasmic reticulum (ER) stress has been reported to play a role in the pathogenesis of intrauterine growth retardation and preeclampsia, especially implantation failure. Although in vitro ER stress studies in human trophoblast cell line have been conducted in recent years, the influence of Thapsigargin on intracellular dynamics on calcium homeostasis has not been proven. Here, the effects of ER stress and impaired calcium homeostasis on apoptosis, autophagy, cytoskeleton, hypoxia, and adhesion molecules in 2D and spheroid cultures of human trophectoderm cells were investigated at gene expression and protein levels. Thapsigargin caused ER stress by increasing GRP78 gene expression and protein levels. Human trophectoderm cells displayed different characterization properties in 2D and spheroids. While it moves in the pathway of EIF2A and IRE1A mechanisms in 2D, it proceeds in the pathway of EIF2A and ATF6 mechanisms in spheroids and triggers different responses in survival and programmed cell death mechanisms such as apoptosis and autophagy. This led to changes in the cytoskeleton, cell adhesion molecules and cell-cell interactions by affecting the hypoxia mechanism.