Gene transfection can be enhanced by heat treatment. In this study, we examined the enhancement mechanism. Heat treatment before transfection showed higher enhancement than during or after the transfection. The heat treatment enhanced the total uptake of plasmid DNA, and increased caveolar endocytosis. As caveolar endocytosis is advantageous for escaping from lysosomal digestion, the amount of intact DNA may increase, resulting in enhanced gene expression.