This study used Radix astragali (RA) as the medium for culturing Cordyceps militaris to investigate the anti-tumor activity of the fermentation broth. It was found that the product from culturing C. militaris in RA medium had a better anti-tumor activity than that culturing in synthetic medium. The fermentation broth inhibited the growth of four tumor cells including human gastric cancer AGS cells, human breast cancer MCF-7 cells, human hepatocellular carcinoma Hep G2 cells and murine colorectal adenocarcinoma CT26 cells with IC(50) 465 mu g/mL, 37 mu g/mL, 25 mu g/mL, and 20 mu g/mL, respectively. Although cordycepin was the major bioactive component with the strongest anti-tumor activity in the fermentation broth of C militaris in RA medium, there were other constituents which enhanced the anti-tumor activity of fermentation broth synergistically. To validate the anti-tumor activity, the BALC/c mice were implanted with CT26 cells and then fed with various dosages of the fermentation product. It was found that 20 mg/kg body weight (BW)/day group had no significant anti-tumor activity as compared to the control group. The dosage of 100 mg/kg BW/day and 200 mg/kg BW/day group inhibited the tumor volume by 43.81% and 48.89%. Tumor weight was also reduced by 31.21% and 39.48% compared to the control group. Besides, the fermentation broth had low cytotoxicity against primary rat hepatocytes, and did not impose serious side effect on the vital organs of the mice as compared to the chemotherapeutic drug 5-FU. (c) 2007 Elsevier Ltd. All rights reserved.