A synergistic approach by the combination of magnetic nanoparticles with an alternating magnetic field for transdermal drug delivery was investigated. Methotrexate-loaded silk fibroin magnetic nanoparticles were prepared using suspension-enhanced dispersion by supercritical CO2. The physiochemical properties of the magnetic nanoparticles were characterized. In vitro studies on drug permeation across skin were performed under different magnetic fields in comparison with passive diffusion. The permeation flux enhancement factor was found to increase under a stationary magnetic field, while an alternating magnetic field enhanced drug permeation more effectively; the combination of stationary and alternating magnetic fields, which has a massage-like effect on the skin, achieved the best result. The mechanistic studies using attenuated total reflection Fourier-transform infrared spectroscopy demonstrate that an alternating magnetic field can change the ordered structure of the stratum corneum lipid bilayers from the gel to the lipid-crystalline state, which can increase the fluidity of the stratum corneum lipids, thus enhancing skin penetration. Compared with the other groups, the fluorescence signal with a bigger area detected in deeper regions of the skin also reveals that the simulated massage could enhance the drug permeation across the skin by increasing the follicular transport. The combination of magnetic nanoparticles with stationary/alternating magnetic fields has potential for effective massage-like transdermal drug delivery.