Introduction: Rapamycin (Rapa) is an immunosuppressive macrolide that inhibits the mechanistic target of rapamycin (mTOR) activity. Thanks to its anti-proliferative effects towards different cell types, including keratinocytes and T cells, Rapa shows promise in the treatment of skin diseases characterized by cell hyperproliferation. However, Rapa skin penetration is limited due to its lipophilic nature (log P = 4.3) and high molecular weight (MW = 914 g/mol). In previous studies, new microenvironment-sensitive core multishell (CMS) nanocarriers capable of sensing the redox state of inflamed skin were developed as more efficient and selective vehicles for macrolide delivery to inflamed skin. Methods: In this study, we tested such redox-sensitive CMS nanocarriers using an inflammatory skin model based on human skin explants co-cultured with Jurkat T cells. Serine protease (SP) was applied on skin surface to induce skin barrier impairment and oxidative stress, whereas phytohaemagglutinin (PHA), IL-17A, and IL-22 were used to activate Jurkat cells. Activation markers, such as CD45 and CD69, phosphorylated ribosomal protein S6 (pRP-S6), and IL-2 release were monitored in activated T cells, whereas pro-inflammatory cytokines were measured in skin extracts and culture medium. Results: We found that alteration of skin barrier proteins corneodesmosin (CDSN), occludin (Occl), and zonula occludens-1 (ZO-1) as well as oxidation-induced decrease of free thiol groups occurred upon SP-treatment. All Rapa formulations exerted inhibitory effects on T cells after penetration across ex vivo skin. No effects on skin inflammatory markers were detected. The superiority of the oxidative-sensitive CMS nanocarriers over the other formulations was observed with regard to drug delivery as well as downregulation of IL-2 release. Conclusion: Overall, our results demonstrate that nanocarriers addressing features of diseased skin are promising approaches to improve the topical delivery of macrolide drugs.