Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.