Mitochondria-targeted sonodynamic therapy (SDT) is a promising strategy to inhibit tumor growth and activate the anti-tumor immune responses. Identifying the mechanisms underlying mitochondria-targeted SDT, further optimizing its efficacy, and developing novel sonosensitizer carriers with good biocompatibility pose major challenges to the clinical practice of SDT. In this study, we investigated the mechanisms of mitochondria-targeted SDT and demonstrated that it suppressed the mitochondrial electron transport chain (ETC) in pancreatic cancer cells through RNA-sequencing analysis. Based on these findings, we constructed the functional lipid droplets (LDs) (CPI-613/IR780@LDs), which combined mitochondria-targeted SDT with the tricarboxylic acid (TCA) cycle inhibitor CPI-613. CPI-613/IR780@LDs synergistically inhibited the TCA cycle and the ETC of mitochondrial aerobic respiration to reduce oxygen consumption and increase reactive oxygen species (ROS) generation at the tumor site, thus enhancing the efficacy of SDT in hypoxic pancreatic cancer. Moreover, the combination of mitochondria-targeted SDT and anti-PD-1 antibody exhibited excellent tumor inhibition and activated anti-tumor immune responses by increasing tumor-infiltrating CD8⁺ T cells and reducing regulatory T cells, synergistically arresting the growth of both primary and metastatic pancreatic tumors. Meanwhile, lipid droplets are cell-derived biological carriers with natural mitochondrial targeting ability and can achieve efficient hydrophobic drug loading through active phagocytosis. Therefore, the functional lipid droplet-based SDT combined with anti-PD-1 antibody holds great potential in the clinical treatment of hypoxic pancreatic cancer.