In this study, we used behavioral and biochemical methods to investigate the effects of Hemerocallis flava (Liliaceae) (abbreviated as HF) on motor activity and the concentration of monoamines in rats. The water fraction of the resuspended HF extract was most active in reducing the motility in rats. The water fraction of the HF extract enhanced the reduction of locomotor activity produced by alpha-methyl-p-tyrosine and 5-hydroxytryptophan, but it reduced the increase of locomotor activity produced by L-dopa plus benserazide and p-chlorophenylalanine. Furthermore, the water fraction of the HF extract significantly decreased the concentration of norepinepherine in the cortex and the concentration of dopamine and serotonin in the brain stem. It also increased the concentration of vanilylmandelic acid in the cortex, homovanillic acid and 5-hydroxyindole-acetic acid in the brain stem. These results suggest that the reduction of locomotor activity produced by the water fraction of HF extract may be related to the decrease in the concentration of norepinepherine in the cortex and the concentration of dopamine and serotonin in brain stem.