An aqueous extract of Simarouba amara was studied for its activity on the differentiation of human skin keratinocytes. Submerged and air-exposed treated keratinocyte cultures displayed a more highly differentiated histoarchitecture, with presence of ultrastructural differentiated elements, than untreated controls. Immunohistochemistry of involucrin and activation of transglutaminase activity provided further evidence for the increase in corneocyte envelope formation observed ultrastructurally. Lipid analysis of air-exposed cultures revealed an increase in the cholesterol sulphate, cholesterol and ceramide contents. After 4 weeks of treatment on the hemiface of volunteers, the capacitance and transepidermal water loss evaluation revealed the potential interest of this extract for improvement of skin hydration. Electron microscopic examination of the corneocyte envelope on tape strips confirmed its actions. Taken together these data demonstrated that an aqueous extract of S. amara increases human keratinocyte differentiation.