Recently, we reported the accelerated gene transfection efficiency of laminin-derived AG73-peptide-labeled polyethylene glycol-modified liposomes (AG73-PEG liposomes) and cell penetrating TAT-peptide labeled PEG liposomes using PEG-modified liposomes, which trap echo-contrast gas, "Bubble liposomes" (BLs), and ultrasound (US) exposure. BLs and US exposure were reported to enhance the endosomal escape of AG73-PEG liposomes, thereby leading to increased gene expression. However, the mechanism behind the effect of BLs and US exposure on endosomes is not well understood. US exposure was reported to induce an influx of calcium ions (Ca²⁺) by enhancing permeability of the cell membrane. Therefore, we examined the effect of Ca²⁺ on the endosomal escape and transfection efficiency of AG73-PEG liposomes, which were previously enhanced by BLs and US exposure. For cells treated with EGTA, the endosomal escape and gene expression of AG73-PEG liposomes were not enhanced by BLs and US exposure. Similarly, transfection efficiency of the AG73-PEG liposomes in ATP-depleted cells was not enhanced. Our results suggest that Ca²⁺ and ATP are necessary for the enhanced endosomal escape and gene expression of AG73-PEG liposomes by BLs and US exposure. These findings may contribute to the development of useful techniques to improve endosomal escape and achieve efficient gene transfection.