Glucan has been shown to have a significant role in the activation of the immune system, including increased activity of macrophages and so on. Sparassis latifolia (formerly S. crispa) is an edible mushroom abundant in dietary fiber and widely known to contain high levels of β-glucan. In the present study, fermentation broths containing soluble β-glucan were prepared by fermentation with mushrooms with four Lactobacillus species (L. plantarum subsp. Plantarum, L. acidophilus, L. helveticus, and L. delbrueckii subsp. Bulgaricus). After culturing four Lactobacillus spp. in MRS broth, each Lactobacillus was inoculated into MRS broth containing S. latifolia powder 5% (w/v) at 37oC in an anaerobic incubator for five days. It showed that the β-glucan contents were different in each fermentation sample. The suitable conditions for the preparation of mushroom fermentation broths were investigated and discussed.