Latanoprost (LAT) has been shown to have a hypertrichotic effect, which makes it a promising candidate for alopecia treatments. For the first time, LAT has been encapsulated in nanotransfersomes in order to increase its efficacy. Ex vivo skin biodistribution was studied by confocal laser microscopy both in human scalp and pig skin. Results showed that nanotransfersomes increase the penetration of two different fluorochromes, with similar patterns in both species, compared with fluorochrome solutions containing no nanotransfersomes. Nanotransfersomes were stable under accelerated conditions (40 °C/75% RH) and long-term conditions (25 °C/60% RH) for up to 1 year, with no differences in vesicle size and polydispersity when LAT was loaded. Nanotransfersomes increased the LAT cell proliferation effect in HaCaT cell via MAPK signaling pathway. Collectively, our results demonstrate LAT-nanotransfersomes formulation could be a promising therapy for hair growth disorders.