Chlorpyrifos (CPF) is an organophosphorus pesticide that is widely used in agricultural production and residential environments worldwide. In this study, we determined the harmful effects and toxicological mechanism of CPF in porcine trophectoderm (pTr) cells and the placenta of female mice during pregnancy. The findings revealed that CPF significantly decreased cell viability and increased intracellular lactate dehydrogenase (LDH) release in pTr cells. Similarly, CPF induced reproductive toxicity in pregnant maternal mice, including decreased maternal, fetal, and placental weights. Moreover, following CPF treatment, pTr cells and the placenta of female mice showed significant apoptosis. JC-1 staining and flow cytometry analysis also revealed that the mitochondrial membrane potential (MMP) of pTr cells treated with CPF was significantly depolarized. Additionally, CPF can induce an increase in reactive oxygen species (ROS) and barrier dysfunction in pTr cells and the placenta of female mice. We further verified that CPF-induced mitochondrial apoptosis is mediated by the MAPK signaling pathway, as shown by using of small molecular inhibitors of related proteins. Also, CPF-induced oxidative stress, barrier dysfunction, and mitochondrial apoptosis in pTr cells were alleviated by U0126, an inhibitor of the ERK/MAPK signaling pathway. These findings suggested that exposure to CPF in early pregnancy might be a potential risk fator affecting placental formation and function in humans and animals.