Many studies have shown that Bupleurum falcatum (BF), which is widely used in the treatment of various psychosomatic diseases in traditional Oriental medicine, is an effective therapeutic intervention for memory impairment. The purpose of this study was to examine the effect of BF on stress-induced alterations in learning and memory in rats using the Morris water maze (MWM) and elevated plus maze (EPM) behavioral tests. In addition, we examined the effects of BF treatment on the cholinergic system, as indicated by changes in neuronal choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) immunoreactivity in the hippocampus. BF (150, 300, or 600 mg/kg) was administered orally 30 min before exposure to repeated immobilization (IMO) stress (4 h/d for 14 d). The BF treatment produced a significant improvement in escape latency (time required to find the platform) in the MWM, and it also produced an anxiolytic-like effect in the EPM. Consistent with the behavioral data, BF treatment significantly attenuated the IMO stress-induced loss of cholinergic immunoreactivity in the hippocampus. These findings indicate that BF has a protective effect against repeated IMO stress-induced neuronal and cognitive impairments, and they suggest that BF may be useful in the treatment of stress-induced memory impairment.