We previously found that 1% minoxidil (MXD) nanoparticles prepared using a bead mill method led to an increase I n hair follicle delivery and hair growth in C57BL/6 mice. In the present study, we designed a nanoparticle formulation containing 5% MXD (MXD-NPs) using the bead mill method and investigated the hair-growth effect of MXD-NPs and a commercially available MXD solution (CA-MXD). Hair growth and in vivo permeation studies were conducted using C57BL/6 mice. Moreover, we examined the MXD contents in the upper (hair bulge) and the lower hair follicle (hair bulb) and observed the hair follicle epithelial stem cells (HFSC) by immunohistochemical staining using the CD200 antibody. The mean particle size of the MXD in the MXD-NPs was 139.8 nm ± 8.9 nm. The hair-growth effect of the MXD-NPs was higher than that of CA-MXD, and the MXD content in the hair bulge of mice treated with MXD-NPs was 7.4-fold that of the mice treated with CA-MXD. In addition, the activation of HFSC was observed around the bulge in the MXD-NPs-treated mice. We showed that MXD-NPs enable the accumulation of MXD in the upper hair follicles more efficiently than CA-MXD, leading the activation of HFSC and the hair growth.