Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutaneous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system.