Lentinula edodes is an edible mushroom that contains a β-glucan called lentinan, which has antitumor and immune-enhancing properties. In the present study, the β-glucan contents of L. edodes mushrooms cultivated on sawdust with different nutritional supplements and fruiting temperatures were measured using a commercial β-glucan assay kit purchased from Megazyme (Bray, Ireland). The weight loss of sawdust media and the yield of fruiting bodies showed similar trends, but the yield was more closely associated with the nutritional supplements used than the weight loss of sawdust media was. The β-glucan contents of L. edodes were 39.5-42.1%, except in the bean curd refuse + CaCl2 supplementation group (50.4%). Furthermore, the β-glucan content decreased with increasing temperatures and was 42.4% at a low fruiting temperature.