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ETHNOPHARMACOLOGICAL RELEVANCE In traditional Chinese medicine (TCM), honey has been used as an additive in the heat-processing of herbal medicines to enhance their immunostimulatory activities. AIM OF THE STUDY We investigated the immunostimulatory activity of heated honey in vitro and in vivo. MATERIALS AND METHODS For the in vitro study, we compared the differences among the inducible effects of honey subjected to various heating conditions on granulocyte colony-stimulating factor (G-CSF) secretion from the cultured enterocytes and investigated the active ingredient. For the in vivo study, we conducted a survival test of mice infected by Streptococcus pyogenes with and without oral administration of heated honey. RESULTS We found that heating the honey induced the appearance of G-CSF secretions from the cultured enterocytes, and that this appearance depended on the heating temperature and time. No G-CSF secretions appeared when honey was not heated. Mice infected with Streptococcus pyogenes that were fed heated honey revealed prolonged survival. The active ingredient in heated honey was a high-molecular compound with about 730 kDa. When this compound was hydrolyzed, galactose, glucose, rhamnose, α-ribofuranose β-ribofuranose 1,5':1',5-dianhydride, and 5-hydroxymethylfurfural were generated. CONCLUSIONS Heated honey reveals immunostimulatory activity both in vitro and in vivo. These results support the scientific evidences of the TCM theory.