<p>Preeclampsia (PE), a severe pregnancy complication, arises from placental hypoxia-induced mitochondrial and endoplasmic reticulum (ER) oxidative stress, contributing to inadequate spiral artery remodeling and endothelial dysfunction. Calpastatin, a mitochondrial protective protein, mitigates oxidative stress-related pathologies, but its role in PE remains unclear. This study investigated the effects of Calpastatin on trophoblast cellular proliferation, migration, invasion, apoptosis, and the expression of autophagy protein (PINK1), mitochondrial dynamics protein (Mfn2), ER stress protein (GRP78), ATP, Ca²⁺, and mitochondrial membrane potential under hypoxia using transfected HTR8-SVneo cells. Calpastatin overexpression significantly enhanced proliferation, migration, and invasion while reducing apoptosis (<i>P</i> &lt; 0.05); knockdown inversely affected these parameters under normoxic conditions. Under hypoxia, overexpression further amplified proliferation and migration (<i>P</i> &lt; 0.01), whereas knockdown reduced migration at 48&#xa0;h (<i>P</i> = 0.04) but not proliferation. Invasion decreased and apoptosis increased in both groups (<i>P</i> &lt; 0.05). Calpastatin overexpression upregulated PINK1, downregulated Mfn2/GRP78, increased ATP and mitochondrial membrane potential, and reduced Ca²⁺. Conversely, knockdown suppressed Pink1/Parkin, elevated Mfn2/Drp1/GRP78, decreased ATP, and increased Ca²⁺ and mitochondrial depolarization (<i>P</i> &lt; 0.05). These findings demonstrate calpastatin promotes trophoblast function by maintaining mitochondrial-ER contact sites stability and ATP production, Ca²⁺ homeostasis, and mitophagy mechanism, suggesting its critical role in PE pathogenesis.</p>