Laser treatment using low fluence for melasma was previously introduced to overcome postinflammatory hypermelanosis after Q-switched laser therapy. However, research on the mechanism of this treatment is very limited. In this study, a collimated low fluence 1064 nm Q-switched Nd:YAG laser with a pulse width of <7 ns was applied using top-hat beam mode. The aim of this study was to investigate the mode of action of this laser treatment through electron microscopy. The effectiveness of this treatment was confirmed by clinical photos, melasma area and severity index and spectrophotometer. To understand the mode of action, the three-dimensional structure of melanocytes in the epidermis was analyzed using serial images acquired by a 3VIEW surface block face scanning electron microscope. In the epidermis, after laser treatment, fewer dendrites in the melanocytes were observed compared with pretreatment. In addition, ultrastructural changes in the melanosome were studied using transmission electron microscopy, which showed that laser treatment caused selective photothermolysis on Stage IV melanosome. Therefore, this treatment should be regarded as an effective method for treating melasma through subcellular-selective photothermolysis.