In this study, a new heteropolysaccharide, here called L2, was separated from the fruit body of Lentinula edodes. Chemical and physical analyses indicated that L2 has a molecular weight of 26 KDa and consists of glucose (87.5%), galactose (9.6%), and arabinose (2.8%), but it does not possess a triple-helical conformation. Stimulation of the production of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in RAW 264.7 cells showed that L2 has significant immune activities involving toll-like receptor 2 (TLR2). Further studies demonstrated that the activities of L2 exhibited high stability in wide range of pH from 4.0 to 10.0, when the thermal processing temperature was below 121 °C. Our findings revealed that a new heteropolysaccharide without triple-helical conformation from Lentinula edodes shows immuno-stimulating activities involving TLR2 at modest pH and thermal processing conditions, which enable it to act as an active component in foods.