Effects of culture temperature ranging from 20 to 34°C on cell growth, polysaccharide biosynthesis and the bioactivity of polysaccharides of Agaricus blazei were evaluated via eight batch cultures in steps of 2°C in a stirred tank bioreactor. Results indicated that the optimal temperature for the biomass was 28°C with a cell yield of 780mgg-1, while that for polysaccharide formation was 30°C with a product yield of 230mgg-1. Both the β-glucan content and average molecular weight of the polysaccharides from different temperature-controlled cultures were closely correlated with their tumour necrosis factor-α (TNF-α) release capability on macrophage cells. The polysaccharides from the low temperature range (20-24°C) not only had higher relative content of β-glucan and average molecular weight but also exhibited higher bioactivity compared with those from the high temperature range (30-34°C). The optimal temperature for the production of bioactive polysaccharides of A. blazei was 24°C, at which their relatively high molecular weight, β-glucan content and TNF-α release capability on macrophage cells were 1650 kDa, 188 mgg-1 and 1560 pg per 5 × 104 cells respectively.