Glucuronoxylomannan (AAPS) from the edible wood ear mushroom Auricularia auricula-judae has been demonstrated to exhibit immunostimulatory properties through its binding to TLR4. However, the mechanisms of immune modulation by AAPS in mammalian cells remains unclear. In the present study, we demonstrated that AAPS induced immunostimulatory effects were regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α and NF-κB. AAPS remarkably increased the phagocytosis and bactericidal activity of macrophages. In lipopolysaccharide-activated macrophages, AAPS induced endotoxin tolerance like effect characterized by the downregulation of nitric oxide, interleukin-6 and TNF-α via the downregulation of NF-κB activation. Our findings provide firm scientific evidences for the immunoenhancing properties of wood ear mushroom, and the potential of AAPS to be strong candidates for the development of new carbohydrate-based nutraceutical supplements in the management of immunity related disorders in the future.