We have been developing a novel compaction method of plasmid DNA using high pressure technology, and previously found that the size of the plasmid DNA was decreased with increasing the pressurizing-strength and time. In the present study, we investigated the tertiary structural change and the expression behavior of the pressure-compacted plasmid DNA in cell in vitro. When the pressure-compacted plasmid DNA was reacted with restriction enzyme (EcoRI), a large amount of the EcoRI was required to cleave the pressure-compacted plasmid DNA than the non-pressurized plasmid DNA, suggesting that the structural change of plasmid DNA was induced by the pressurization. The expression of the pressure-compacted plasmid DNA injected into cells using microinjection method was analyzed. It was clear that the pressure-compacted plasmid DNA could effectively delay gene expression, suggesting that the controlled compaction of plasmid DNA by high pressurization would be regulate the transgene expression.