Lentinula edodes is one of the most widely used medicinal mushrooms which exhibit significant antitumor activity. It is mostly studied and several studies have showed that its polysaccharides play critical roles in the antitumor effects. In this study, we determine the antitumor activity of Lentinula edodes C91-3 mycelia fermentation protein (LFP91-3) on S180tumor cells in vivo as well as in vitro. The results showed that LFP91-3 inhibited the growth of S180 tumor cells in vitro, and the mechanism may be related to apoptosis induced by LFP91-3. In vivo, LFP91-3 treatment significantly prolonged life span in S180 tumor-bearing mice. LFP91-3 can be used as an important antitumor element in Lentinula edodes C91-3 mycelia fermentative liquid. Key words: Lentinula edodes C91-3 fermentation protein, Lentinula edodes, antitumor activity.