β-sitosterol glucoside exists in a variety of plants and have anti-tumor, anti-microbial, and immunomodulatory activities. Mast cells and eosinophils play important roles in a variety of inflammatory diseases, specifically asthma and atopic dermatitis. In the present study, we used lactose-β-sitosterol (L-BS) and investigated the effect of L-BS on inflammatory responses of the human mast cell line, HMC-1 and the human eosinophilic leukemia cell line, EoL-1. In HMC-1 cells, L-BS significantly inhibited cell migration in response to stem cell factor without cytotoxicity. However, the mRNA expression of CC chemokine receptors (CCRs), including CCR1-5, were not altered after L-BS treatment in HMC-1 cells. LPS-induced IL-4 production was also suppressed by L-BS in a dose-dependent manner. In EoL-1 cells, the concentration ranging from 0.1 μM to 10 μM of L-BS had no cytotoxicity and had no effect on mRNA expression of major protein-mediators derived from activated eosinophils. However, 100 μM of L-BS induced the apoptosis of EoL-1 cells in a time-dependent manner. This finding indicates the possibility of L-BS as a potential therapeutic molecule in inflammatory diseases and may contribute to the need to improve current therapeutic drugs.