Topical isotretinoin is commonly used to treat acne. However, topical isotretinoin has side effects and can hardly permeate through the stratum corneum, the most important skin barrier. Therefore, this study aimed to demonstrate the efficacy of nanoparticles as stable carriers with great curative effects, low side effects, and strong transdermal ability. In a rabbit model of hyperkeratinization, STCM-ATRA-NPs showed significant therapeutic efficacy. By contrast, negative therapeutic efficacy was observed in a golden hamster model of hyper sebum production. Scanning electron microscopy and Fourier transform infrared spectral analyses showed that nanoparticles could penetrate the stratum corneum. Western blotting demonstrated that the nanoparticles could enhance the transdermal efficacy of isotretinoin by reducing the effect of keratin and tight junction proteins. Further, nanoparticles enhanced endocytosis, thereby promoting drug penetration and absorption into the skin. STCM-ATRA-NPs were demonstrated to control isotretinoin release, reducing its side effects, and efficiently permeating through the skin by reducing the effect of keratin and tight junction proteins and enhancing endocytosis.