Bacteria continuously change their genetic characteristics to adapt to the changing environment by means of horizontal gene transfer. Although three conventional mechanisms of horizontal gene transfer are well known (transformation, transduction, and conjugation), new variations of these mechanisms have also been described. We previously reported that DNase-sensitive cell-to-cell transfer of non-conjugative plasmids, termed as "cell-to-cell transformation," occurs between the cells of two Escherichia coli strains in a co-culture. In this study, to further investigate the mechanism of cell-to-cell transformation, we constructed a new experimental system for cell-to-cell transformation. By using this system, we found that high temperatures of approximately 41ºC-45 °C significantly promote cell-to-cell plasmid transformation. This transfer was much more frequent in solid-air biofilms than in liquid culture, suggesting an importance of biofilm environment. Plasmid transfer frequency reached over 10-7/cell under the optimal strain-plasmid combination and conditions tested. DNase sensitivity test and plasmid isolation from the transformants confirmed the horizontal transfer of full-length plasmids via transformation. Comparative natural transformation experiments, which used similar strains and plasmids under equivalent culture conditions, revealed that cell-to-cell transformation occurs approximately 103 times more frequently than natural transformation, indicating the uniqueness and effectiveness of the cell-to-cell transformation mechanism. As temperatures of approximately 41ºC-45 °C are common in the avian intestines and under some other environmental situations, the phenomenon demonstrated here can occur efficiently in such locations. To the best of our knowledge, this is the first study to demonstrate the enhancing effect of high temperatures on cell-to-cell plasmid transformation in E. coli.