Objectives Heat treatment as a physical method could increase the cellular uptake of nucleic acids. In this study, the effects of heat shock were evaluated to enhance the transfection efficiency of three plasmid DNAs into HeLa and TC-1 cancerous, and HEK-293 T and Vero non-cancerous cell lines using lipofectamine 2000 reagent. Methods Two methods of cell- and DNA-based heat treatment were used. Heating DNA solution was performed at 94 degrees C for 5, 10 and 15 min, and also 72 degrees C for 30, 60 and 120 min, individually. Moreover, heating the cells was done by incubation at 42 degrees C for 2 h in different times such as before, during and after DNA transfection. Results Our data showed that the conformation of plasmid DNAs was changed at different temperatures with increasing time. The heat-treated plasmid DNAs (94 degrees C for 10 min or 72 degrees C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs (p < 0.05). Furthermore, heat treatment of cells before and during the transfection was higher than untreated cells (p < 0.01). Our results demonstrated that DNA transfection efficiency in cancerous cells was less than non-cancerous cells (p < 0.01). Conclusion Generally, these findings showed that transfection mediated by thermal stimulation could enhance gene transfection in mammalian cell lines.