Hangeshashinto (HST) has been used as an herbal formula to treat inflammatory ulcerative gut diseases complicated with psychoneurosis in Japanese traditional Kampo medicine. In our previous study, we revealed that HST improved rat inflammatory colitic symptoms induced by the intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The aim of the present study is to clarify the anxiolytic effect of HST in the same model. The anxiety-like behavior was measured using an elevated plus maze (EPM). TNBS-treatment reduced the exploring time spent on the open arms of EPM, suggesting that as anxiety-like behavior was observed. This behavior was significantly improved by daily oral administration of HST. To identify the active constituents for this anxiolytic effect, berberine (BE), baicalin (BA), glycyrrhizin (GL) or saponin fraction of ginsenosides (GS), or their combinations were tested. Oral treatment with these four constituents significantly ameliorated the exploration time spent on open arms, although no effects were found in the treatment with each of BA, BE, GL or GS. Among the combinations in these 4 constituents, the combination of BA and BE exerted the anxiolytic effect, however, the combination of GL and GS, which is the active combination to improve colitic symptoms (damage score, colonic wet weight and ulcerative area) in our previous study (Kawashima et al. Biol. Pharm. Bull. 27, 1599, 2004), did not show any improvement. These results suggest that HST would have anxiolytic effect, which would be partially derived from the combination of BA and BE, and HST ameliorated the colitis, which would be partially derived from the combination of GL and GS, in TNBS-colitis model.