The potential for low-frequency ultrasound facilitated topical transport of Cyclosporin A was investigated using rat skin. Studies of intensity and exposure time acting on the deposition of Cyclosporin A into deeper skin of in vitro sonophoresis were performed. Low-frequency ultrasound increased the amount of Cyclosporin A retained in the skin only seven times than the passive diffusion. Furthermore, we also tested the synergistic effect of ultrasound and other approaches such as chemical enhancers and electroporation on topical drug delivery of Cyclosporin A. We found that the efficacy of low-frequency ultrasound in enhancing topical delivery could be further increased by pretreatment of skin with chemical enhancers, such as laurocapram (Azone) and sodium lauryl sulfate (SLS). Meanwhile only a small amount was seen to across the full skin into the receiver compartment. Trimodality treatment comprising of pretreatment with Azone+ultrasound in combination followed by electroporation was not effective in enhancing the topical delivery of Cyclosporin A. However, this combination strategy increased the penetration of Cyclosporin A through rat skin by order of 15. The histopathological findings revealed that there was almost no change observed in the structure of skin after ultrasound or combination with ultrasound and enhancers as compared with the control group. In general, the enhanced skin accumulation of Cyclosporin A by the combination of low-frequency ultrasound and chemical enhancers could help significantly to optimize the targeting of the drug without of a concomitant increase of the systemic side effects.