Natural killer (NK) cells play a crucial role in antitumor immunity, but their functionality is often impaired in acidic tumor microenvironment (TME). This study demonstrates that NK cells primed with 25 kDa branched polyethylenimine (25KbPEI), termed Chem_NK, retain antitumor activity under acidic conditions (pH6.0) comparable to control NK (C_NK) in neutral conditions (pH7.5). In contrast, the antitumor activity of C_NK significantly declines at pH6.0. Unlike C_NK, Chem_NK preserve mitochondrial function, as evidenced by sustained oxidative phosphorylation under acidic conditions. Mitochondrial imaging confirmed Chem_NK’s resistance to acidosis-induced fragmentation. Furthermore, phosphorylation analysis of mitochondrial fission protein dynamin-related protein 1 (DRP1) revealed that Chem_NK exhibited decreased phosphorylation at the activation site (S616) and increased phosphorylation at the inhibitory site (S637), indicating reduced susceptibility to mitochondrial fission under acidic conditions. Chem_NK also showed elevated activity of protein kinase A (PKA), an upstream regulator of DRP1^S637 phosphorylation. Pharmacological inhibition of PKA impaired both migration and cytotoxicity, which were otherwise sustained at levels comparable to C_NK in neutral conditions. These findings underscore that Chem_NK preserve mitochondrial function via the PKA-DRP1 axis, enabling enhanced antitumor activity under acidic stress and highlighting a promising strategy to improve NK cell-based immunotherapy.