<p>Cellular metabolic reprogramming is intimately linked to various physiological and pathological processes. For instance, calcium (Ca²⁺)-mediated signaling pathways are essential for maintaining the homeostasis of critical cellular organelles. Stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) is a primary pathway for Ca²⁺ influx in non-excitable cells. This study aims to elucidate the role of STIM1 in podocyte injury. An STIM1 eukaryotic overexpression plasmid (p-STIM1) and small interfering RNA (si-STIM1) were constructed and separately transfected into mouse podocytes (MPC5). Flow cytometry was used to assess apoptotic rates, Fluo-3/AM calcium imaging to measure intracellular Ca²⁺ levels, and Western blotting to analyze the expression of endoplasmic reticulum stress (ERS)-related proteins. Additionally, mitochondrial morphology, membrane potential (MMP), reactive oxygen species (ROS) levels, and mitochondrial DNA (mtDNA) copy numbers were evaluated. Compared to STIM1 deficiency, STIM1 overexpression led to a marked increase in the apoptotic rate of Adriamycin-induced injured podocytes in vitro. This was associated with a significant rise in intracellular Ca²⁺ concentration and upregulation of ERS-related proteins, including GRP78, GRP94, and CHOP. Mitochondria displayed pronounced swelling and vacuole-like changes, a notable reduction in MMP, elevated ROS levels, and a decrease in mtDNA copies. STIM1 exacerbates podocyte injury by promoting intracellular Ca²⁺ influx, intensifying ERS, and inducing significant morphological and functional mitochondrial alterations. These findings suggest that targeting STIM1-mediated pathways could be a potential therapeutic strategy for podocyte-related kidney diseases.</p>