One of the major female sex hormones, estrogen, can influence a variety of mental states. Individuals with multiple sclerosis (MS) often suffer from mental health issues, which are correlated with the pathology of gray matter. In this study, we aimed to elucidate the validity of phytoestrogen genistein (GEN) for treating the gray matter lesions in MS using the mouse model of cuprizone (CPZ)-induced demyelination. First, we confirmed that 5-week 0.2% CPZ intoxication induced demyelination in the hippocampus, and that myelination was successfully recovered by GEN. Loss of mature oligodendrocytes following CPZ intoxication was counteracted by GEN. Neither CPZ nor GEN affected the densities of oligodendrocyte precursor cells and astrocytes. CPZ-induced activation and proliferation of microglia were not inhibited by GEN. Upregulation of gene expression of the pro-inflammatory cytokine, interleukin-1β, in sorted microglia by CPZ was not inhibited by GEN either. However, the expression levels of genes related to phagocytosis, such as cluster of differentiation 68 and lysosomal-associated membrane protein 1, in sorted microglia were elevated by CPZ but lowered by GEN. Notably, physical contact of microglia with myelin was increased by CPZ but decreased by GEN. The expression levels of myelin-related genes, such as myelin basic protein and myelin oligodendrocyte glycoprotein, in the whole hippocampal tissue were decreased by CPZ but recovered by GEN. These results show that GEN may act on mature oligodendrocytes in the hippocampus by promoting their survival and myelin formation, and suggest the therapeutic potential of phytoestrogens for treating MS patients suffering from mental health issues.