Despite their crucial role in antitumor immunity, natural killer (NK) cells often experience metabolic impairments in the acidic tumor microenvironment (TME), leading to functional exhaustion. In this study, we demonstrate that NK cells primed with 25 kDa branched polyethylenimine (Chem_NK) retain robust antitumor activity even under acidic stress conditions. Chem_NK showed significantly prolonged stable contact with target tumor cells at pH 6.0, compared to control NK cells (C_NK). Mechanistically, Chem_NK exhibited enhanced mitochondrial function, characterized by elevated oxidative phosphorylation and mitochondrial membrane potential regardless of pH. Notably, mitochondrial integrity of Chem_NK was preserved under acidosis, in contrast to C_NK, which accumulated DRP1 and exhibited mitochondrial fragmentation. Chem_NK maintained phosphorylation of DRP1 at serine 637 via PKA signaling, conferring resistance to acidosis-induced mitochondrial dysfunction. Additionally, this mitochondrial resilience translated into superior migration and cytotoxic performance under acidic conditions. These findings highlight Chem_NK as a metabolically resilient NK cell population capable of overcoming TME-associated acidosis, and underscore the therapeutic potential of metabolic priming for NK cell-based cancer immunotherapy.