The impacts of microwave assisted-extraction (MAE) on the physicochemical characteristics, functionalities, and digestion behavior of Lactarius volemus (L. volemus) protein isolate (LPIs) was investigated. Compared with the conventional water bath assisted-extraction method (WAE), MAE significantly enhanced the extraction rate of LPIs by 30.00% and 47.98% at 20 and 60 min, respectively. Also, MAE unfolded the spatial structure of LPIs, promoting the transformation from ordered structure to disordered structure, exposing its hydrophobic groups and increasing free sulfhydryl content. In addition, LPIs obtained by MAE showed better solubility, emulsifying and foaming characteristics than that by WAE. MAE method can improve the digestibility and the degree of hydrolysis of LPIs compared to WAE, which were increased by 6.06% and 19.78% after 20 min extraction in the small intestine digestion, respectively. This study can provide a potential strategy to produce L. volemus protein isolate with high efficiency and quality.