Shiitake mushrooms contain lentinan, a polysaccharide that has numerous medicinal benefits. The objective of this research was to determine the impact of various rates of chipped and sawmill bark added to the substrate on lentinan found in shiitake mushrooms. Crude polysaccharide extract (CPE) and lentinan (LTN) content of shiitake mushrooms were significantly different among artificial substrates containing varying percentages of sawdust and chipped bark. The addition of chipped bark content from 0% to 50% increased the CPE and LTN content, but an increase in chipped bark content from 50% to 100% decreased the CPE and LTN content. CPE content in mushrooms was greater when equal proportions of sawdust and bark were used. A 100% heartwood treatment had the least CPE and LTN content.