Using nanoparticle-based drug delivery systems as enhancers is a robust strategy for transdermal deliv-ery; however, the mechanisms by which these systems promote transdermal penetration are still unclear. Here, we fabricated a dual-labeled nano drug delivery system that allows discrete visualization of both the drug and the nanoparticle carrier. To comprehensively examine its potential mechanism, we inves-tigated its effects on human epidermal keratinocyte HaCaT cells, including changes in cell membrane potential, intracellular Ca2+ concentration, and Ca2+-ATPase activity. P-glycoprotein (P-gp) expression in nanoparticle-treated human dermal microvascular endothelial cells was detected by western blotting and immunofluorescence. Furthermore, the transdermal absorption and biodistribution of the dual-labeled nanoparticles were deeply investigated by skin permeability study in vitro and in vivo using fluorescence microscopy and in vivo imaging, respectively. In addition to reducing membrane potential, increasing the intracellular Ca2+ concentration, and decreasing Ca2+-ATPase activity, our results indicate that the dual-labeled nanoparticles can downregulate P-gp to promote transdermal absorption. Fluorescence and in vivo imaging visually demonstrated that the nanoparticle delivery system penetrated into the dermis through the stratum corneum. All these results indicate that this dual-labeled nano delivery system provides a new method for future in-depth visual explorations of transdermal drug delivery mechanisms.