The leaves of Perilla frutescens Britton var. acuta Kudo (Perillae Herba) are found in some traditional oriental herbal medicines that are primarily used to treat affective disorders such as depression and anxiety. The aim of the present study was to identify the bioactive component in Perillae Herba that possesses antidepressive activity. The effects of a water extract of Perillae Herba and six fractions therefrom were evaluated in mice by use of the forced-swimming test. An oral administration of a water extract of Perillae Herba significantly reduced the duration of immobility. Moreover, 50% methanol extract of the water extract of Perillae Herba and its 30% methanol extract also reduced the duration of immobility. Three-dimensional high-performance liquid chromatography and FAB-MS and NMR spectral analysis clearly showed that the extracts with anti-immobility effects contained abundant rosmarinic acid. The oral and intraperitoneal administration of rosmarinic acid significantly reduced the duration of immobility. In contrast, a water extract from another species of Perillae Herba, which contains only low levels of rosmarinic acid, had no anti-immobility effect. These results suggest that rosmarinic acid may be the main component involved in the antidepressive effect of Perillae Herba in the forced-swimming test. Thus it may be a novel antidepressive substance.