β-sitosterol, one of phytosterols, exhibited numerous pharmacological effect including anti-inflammatory, anti-cancer and immune-modulating properties. This study attempted to determine the pharmacological effects of β-sitosterol on atopic dermatitis (AD). We investigated to ascertain the pharmacological effects of β-sitosterol on 2, 4-dinitrochlrobenzene (DNCB)-induced AD symptom and histamine-induced scratching behaviors in mice. Additionally, we evaluated the effects of β-sitosterol on the interleukin (IL)-6 levels in HaCaT cells and skin tissue of AD. The findings of this study demonstrated that β-sitosterol reduced AD clinical symptoms such as eczematous, erythema and dryness and serum histamine and IgE levels in DNCB-induced AD model and histamine-induced scratching behaviors in mice. Additionally, β-sitosterol inhibited the IL-6 expression in AD-like skin lesion and HaCaT cells. Collectively, these findings provide that β-sitosterol could be a therapeutic agent for skin inflammation including AD.