6:2 chloro-polyfluorooctane ether sulfonate (F-53B) is considered neurotoxic, but its mechanisms remain unclear. This study aimed to investigate the toxic effects of F-53B on neuronal cells, focusing on the role of the V-ATPase-AMPK axis in the mechanism of abnormal energy metabolism. Mouse astrocytes (C8-D1A) and human neuroblastoma cells (SH-SY5Y) exposed to F-53B were used as in vitro models. Our findings demonstrated that F-53B inhibited the expression of V-ATPase B2 and reduced V-ATPase activity, leading to an increase in lysosomal pH, decreased expression of TRPML1, and lysosomal Ca