β-glucan is a safe and highly potent biological response modifier that nutritionally activates the immune response through the Macrophage, Dendritic and additional immune cells to yield various therapeutic effects. Shiitake mushrooms (Lentinula edodes) contanining β-glucans may be beneficial for human health; they have been used in the treatment of cancer, hypertension, and high cholesterol levels. The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on β-glucan production in the edible mushroom L. edodes was studied. The cap of the mature mushroom showed the highest β-glucan activity, and β-glucan activity seemed to be influenced somewhat by some well-known inducers or sawdust. In this study, we utilized five strains (JMI 10022, 10036, 10077, 10079, 10080) of L. edodes regardless of origin and growth conditions. This experiment showed that the expression of β-glucan was induced by glucose bond, and increased with the growing of L. edodes. Quantitatively, reverse transcription PCR utilized pairs of primers specific for β-glucan gene expression shows that expressed genes were most commonly indentified during the process of fruiting-body formation. We suggest that the results will provide valuable information to assist L. edodes industry.