This study presents an acoustothermal transfection method that uses acoustic and thermal effects to enhance cell membrane and nuclear envelope permeability, enabling efficient and safe transfection of primary T cells and stem cells. The method leverages surface acoustic waves (SAWs) to generate acoustic energy and thermal energy through viscous damping, which transiently increases cell membrane and nuclear envelope permeability. This allows for the efficient delivery of large plasmids into the nuclei of mesenchymal stem cells (MSCs) with high efficiency (89.6 ± 1.2%). CXCR4-transfected MSCs can efficiently target cerebral ischemia sites in vivo and reduce infarct volume in mice. The acoustothermal transfection method addresses the key challenge of balancing transfection efficiency and cell viability, offering a powerful tool for cellular and gene therapies. The method was validated using MCF-7 cells, MSCs, and primary T cells, showing high transfection efficiency with minimal cell damage. For example, MCF-7 cells showed strong green fluorescence with an efficiency of 96.1 ± 0.5% after transfection. MSCs transfected with CXCR4-EGFP plasmids showed gene expression efficiencies of 89.0 ± 0.9% and 82.6%, while those transfected with BDNF plasmids showed efficiencies of 90.3 ± 2.4% and 86.7%. For T cells, a single type of plasmid expression reached 85.0 ± 0.6% (81.3% with flow cytometry) in 24 hours. The transfected cells could realize therapeutic functionalities in vivo by injecting transfected MSCs into ischemic stroke mouse models. The acoustothermal transfection method was shown to have high efficiency, high throughput, and high biocompatibility when delivering large plasmids into hard-to-transfect cells. The method was also validated for its biocompatibility, with no significant gene mutation or cell death observed. The method was found to be effective in maintaining cell viability and function in vivo, as demonstrated by the successful treatment of cerebral ischemia in mice. The study concludes that the acoustothermal transfection method is a promising approach for gene and cell therapies, offering a robust, scalable, and biocompatible solution for efficient and safe transfection of hard-to-transfect cells.This study presents an acoustothermal transfection method that uses acoustic and thermal effects to enhance cell membrane and nuclear envelope permeability, enabling efficient and safe transfection of primary T cells and stem cells. The method leverages surface acoustic waves (SAWs) to generate acoustic energy and thermal energy through viscous damping, which transiently increases cell membrane and nuclear envelope permeability. This allows for the efficient delivery of large plasmids into the nuclei of mesenchymal stem cells (MSCs) with high efficiency (89.6 ± 1.2%). CXCR4-transfected MSCs can efficiently target cerebral ischemia sites in vivo and reduce infarct volume in mice. The acoustothermal transfection method addresses the key challenge of balancing transfection efficiency and cell viability, offering a powerful tool for cellular and gene therapies. The method was validated using MCF-7 cells, MSCs, and primary T cells, showing high transfection efficiency with minimal cell damage. For example, MCF-7 cells showed strong green fluorescence with an efficiency of 96.1 ± 0.5% after transfection. MSCs transfected with CXCR4-EGFP plasmids showed gene expression efficiencies of 89.0 ± 0.9% and 82.6%, while those transfected with BDNF plasmids showed efficiencies of 90.3 ± 2.4% and 86.7%. For T cells, a single type of plasmid expression reached 85.0 ± 0.6% (81.3% with flow cytometry) in 24 hours. The transfected cells could realize therapeutic functionalities in vivo by injecting transfected MSCs into ischemic stroke mouse models. The acoustothermal transfection method was shown to have high efficiency, high throughput, and high biocompatibility when delivering large plasmids into hard-to-transfect cells. The method was also validated for its biocompatibility, with no significant gene mutation or cell death observed. The method was found to be effective in maintaining cell viability and function in vivo, as demonstrated by the successful treatment of cerebral ischemia in mice. The study concludes that the acoustothermal transfection method is a promising approach for gene and cell therapies, offering a robust, scalable, and biocompatible solution for efficient and safe transfection of hard-to-transfect cells.