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The use of herbicides is a major control method in agriculture. Pendimethalin (PDM) has been increasingly used as a herbicide for approximately 30 years. PDM has been reported to cause various reproductive problems, but its toxicity mechanism in the pre-implantation stage has not been investigated in detail. Herein, we studied the effects of PDM on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells and identified a PDM-mediated anti-proliferative effect in both cell types. PDM exposure generated intracellular ROS, induced excessive Ca2+ influx into mitochondria, and activated MAPK signaling pathway. Ca2+ burden resulted in the dysfunction of mitochondria and eventual disruption of Ca2+ homeostasis. Further, PDM-exposed pTr and pLE cells showed cell cycle arrest and programmed cell death. In addition, a decrease in migration ability and dysregulated expression of genes related to the functioning of pTr and pLE cells was evaluated. This study provides insight into time-dependent transitions within the cell environment after PDM exposure and elucidates a detailed mechanism of induced adverse effects. These results imply that PDM exposure can potentially cause toxic effects on implantation-related process in pigs. Moreover, to the best our knowledge, this is the first study to describe the mechanism by which PDM induces these effects, enhancing our understading of the toxicity of this herbicide.