Methotrexate exhibits poor cutaneous bioavailability when administered topically using conventional vehicles. It is recommended for the treatment of skin-related diseases but its systemic side effects hamper application. The aim of this work was to formulate methotrexate in nanostructured lipid carriers using biocompatible lipids, and to investigate their potential for topical drug delivery. Methotrexate-loaded nanostructured lipid carriers were prepared via a hot ultrasonication method. Nanocarriers were evaluated for size, polydispersity, surface potential and entrapment efficiency. Shape and surface morphology of the produced lipid carriers confirmed their spherical shape. After 10 h ca. 50% of methotrexate was released in vitro from the lipid nanocarriers following Peppas–Korsmeyer release kinetics. Moreover, methotrexate-loaded nanocarriers were stable at least for 3 months and less toxic towards fibroblasts and human keratinocytes than the free drug. Methotrexate within lipid nanocarriers was able to pass the experimental keratinocyte epidermal barrier at a flux rate of 2 μg cm−2 h−1. Results demonstrated that lipid nanocarriers constitute a suitable approach for application of methotrexate in skin-related diseases topical therapy.