The present study examined the stability of a gene in powders prepared with supercritical carbon dioxide (CO₂) from the viewpoints of the ternary structure of DNA and in vivo transfection potential. An aqueous chitosan–pCMV-Luc complex solution containing mannitol was injected into the stream of a supercritical CO₂/ethanol admixture to precipitate a gene powder. The obtained gene powders and gene solutions were placed in stability chambers at 25 or 40°C for 4 weeks. The integrity and transfection potency of the gene were examined by electrophoresis and in vivo pulmonary transfection study in mice. The supercritical CO₂ process decreased the supercoiled DNA during the manufacturing process; however, the decrease in the remaining supercoiled and open circular DNA in the powders during storage was much slower than that in solutions. In addition, the powders had higher transfection potency than the solutions containing the same amount of DNA. The effect of chitosan on the stability of DNA in solutions was not obvious in the solutions but it improved the stability of DNA in powders during manufacturing and storage. Thus, a gene powder with a cationic vector is a promising ready-to-use formulation for inhalation therapy of pulmonary diseases.