OBJECTIVE : Intracerebroventricular (ICV) administration of ultrasound-responsive bubbles and cranial ultrasound irradiation is reported as a transfection system for the cerebroventricular region. This study aimed to characterize the transfection system with respect to transfection efficiency, spatial distribution of transgene expression, and safety. METHODS : Plasmid DNA was transfected to mouse brain by ICV injection of ultrasound-responsive nanobubbles, followed by ultrasound irradiation to brain. Spatial distribution of transgene expression in the cerebroventricular region was investigated using multicolor deep imaging. RESULTS : This transfection system efficiently transferred the transgene to the choroid plexus with no morphological change or cerebral hemorrhage. Moreover, sustained secretion of transgenic protein was achieved by transferring the transgene encoding the secretable protein. CONCLUSIONS : We successfully developed an ultrasound-responsive nanobubbles-mediated method for gene transfection into the cerebroventricular region via ICV administration in mice.