The present study aimed to evaluate the influence of ultrasound assisted HO/ascorbic acid reaction on the structural characteristic and immunostimulatory activity of a β-D-glucan isolated from D. indusiata, so as to reveal its potential structure-immunostimulatory activity relationship. A purified β-D-glucan, named as DP, was quickly isolated from D. indusiata, and further identified as a 1,3-β-D-glucan with 1,6-β-D-Glcp as branched chains, which exhibited a rigid rod chain conformation in 0.9 % (w/v) of NaCl solution. Furthermore, results showed that the primary structure of DP was overall stable after the degradation by ultrasound assisted HO/ascorbic acid reaction. However, the molar mass and chain conformation of DP obviously changed. In addition, DP and its degraded products exerted remarkable immunostimulatory activity in vitro and in vivo, which could activate the nuclear factor-κB (NF-κB) signaling pathway through toll-like receptor 4 (TLR4). Indeed, the immunostimulatory activity of DP was closely-correlated to its molar mass and chain conformation. An appropriate degradation of molar mass could promote its immunostimulatory activity. While the transformation of chain conformation from rigid rod to random coil could cause the significant decrease of its immunostimulatory activity. These findings are beneficial to better understanding the structure-immunostimulatory activity relationship of β-D-glucans from edible mushrooms.