Our laboratory has previously demonstrated that cytoplasmic trafficking and subsequent nuclear entry of nonviral plasmid DNA can be significantly enhanced through the application of cyclic stretch after transfection in vitro. In this study, we show that cyclic stretching of the murine lung using ventilation immediately after endotracheal administration and transthoracic electroporation of plasmid DNA increases exogenous gene expression up to fourfold in mice that were not ventilated after plasmid administration and transfection by electroporation in vivo. This increase is both time and sequence specific (that is, the ventilation must occur immediately after the transfection event). The ventilation-enhanced gene transfer is also amplitude dependent, confirming similar studies completed in vitro, and is mediated, at least in part, through the cytoplasmic tubulin deacetylase, HDAC6. Using immunohistochemistry, we show that this increase in expression is due to an increase in the number of cells expressing the exogenous protein rather than an increase in the amount of protein produced per cell. These studies show the potential mechanical stimulation has in vivo in significantly increasing nonviral DNA gene expression, and may ultimately pave the way for more successful clinical trials using this type of therapy in the future.