To improve 17-α-estradiol targeting in alopecia treatment, a formulation of nanostructured lipid carrier (NLC) prepared by the sonication method was developed. The physicochemical characterization showed an average diameter of 96 ± 15 nm and the average zeta potential of -17 ± 6 mV. In addition, the encapsulation efficiency (EE) of 17-α-estradiol, assessed by ultra-performance liquid chromatography (UPLC) fluorescence, was 99.6 ± 0.3%. Physical stability of NLC stored at room temperature was followed for 42 days through several physicochemical parameters, such as diameter, zeta potential, pH and EE. No changes were observed in the physicochemical parameters during this period, demonstrating the potential of NLC as a delivery system of 17-α-estradiol. NLC dermal penetration, investigated by Franz diffusion cell and confocal microscopy using rhodamine-labeled NLCs, showed an accumulation of NLC in the outer portions of the epidermis, precisely in the hair follicle. The NLCs distribution in the skin suggests that 17-α-estradiol targeting was improved.