Kampo (traditional Japanese herbal) medicines are taken orally due to which the gastric mucosal immune system may act as one of the major targets for the expression of pharmacological activity. The inner surface of the intestinal tract possesses a large area of mucosal membranes, and the intestinal epithelial cells sit at the interface between a lumen and a lymphocyte-rich lamina propria. The cross talk that occurs between these compartments serves to maintain intestinal homeostasis, and the cytokine network plays an important role in the cross talk. In this study, the effect of Hochuekkito (HET), one of Kampo medicines, on cytokine secretion of intestinal epithelial cells was investigated. When murine normal colonic epithelial cell-line MCE301 cells were stimulated with HET, the contents of granulocyte colony-stimulating factor (G-CSF) in the conditioned medium were significantly increased in dose- and time-dependent manners. The enhanced G-CSF gene transcription in MCE301 cells by the stimulation of HET was observed by RT-PCR. The enhanced G-CSF secretion by HET was also observed in C3H/HeJ mice-derived primary cultured colonic epithelial cells. When the HET was fractionated, only the polysaccharide fraction (F-5) enhanced the G-CSF secretion of MCE301 cells, and the activity of F-5 lost after the treatment of periodate that can degrade the carbohydrate moiety. These results suggest that HET enhances secretion of G-CSF from colonic epithelial cells and the polysaccharide is one of the active ingredients of HET. The enhanced G-CSF secretion by HET may partly contribute to the clinically observed various pharmacological activities of HET including immunomodulating activity.