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Background: The 75-kDa glucose-regulated protein (GRP75) plays a crucial role in regulating the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), facilitating the transfer of Ca2+ ions, and is essential for lipid metabolism, mitochondrial function, calcium homeostasis, and apoptosis in mammalian cells. However, the relationship between GRP75 expression and preimplantation embryonic development in pigs remains unknown. Methods: In this study, we investigated whether GRP75 influences ER–mitochondrial junctions and mitochondrial Ca2+ levels in porcine embryos in vitro . We examined the expression of GRP75 at the zygote, cleavage, and blastocyst stages using immunofluorescence staining and western blot analysis. Results: GRP75 fluorescence and mitochondrial Ca2+ levels were significantly lower (p < 0.01) in the blastocysts than in the zygotes. Western blot analysis revealed a decline in the expression of mitochondrial fusion factors mitofusin 2, GRP75, and the mitochondrial calcium uniporter complex MICU1 protein at the blastocyst stage. To investigate the effects of GRP75 on blastocyst developmental competence, porcinespecific GRP75-siRNA (25 nM) was microinjected at the zygote stage. The results showed a significant decrease in the development capacity until the blastocyst stage (Control: 31.2 ± 2.0%, N.C. siRNA (25 nM): 29.8 ± 3.1%, vs. GRP75-siRNA (25 nM): 24.1 ± 1.6%; p < 0.05). GRP75 in the mitochondria and ER-localized GRP75 were both significantly reduced in blastocysts of pigs microinjected with GRP75 siRNA. Along with ER–mitochondrial colocalization, the MAM formation ratio was significantly reduced in the GRP75-siRNA group compared with that in the control (Control: 29.3% vs. GRP75- siRNA (25 nM): 15.7%, p < 0.001). Conclusions: These findings demonstrate that the GRP75-derived MAM region is involved in the development of early embryos in porcine blastocysts.