High glucose can lead to toxicity on islet β cells. The protective effects of a novel Lentinus edodes mycelia polysaccharide (LMP) on INS-1 cells damaged by glucose were investigated. Cell viability, lactate dehydrogenase (LDH) release, cell apoptosis, intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were detected. P38 MAPK, JNKand NF-κB pathways were analyzed to reveal the inhibitory mechanism of LMP on glucose-induced INS-1 cells toxicity. The results showed that LMP could decrease cellular oxidative stress, reduce intracellular ROS levels, decrease MDA content and increase SOD activity. Furthermore, the glucose-induced cell apoptosis in cells were inhibited by regulating the expression of Bax, Bcl-2, cleaved caspase‑3 and cleaved caspase‑1. Cell signaling pathway analysis revealed that LMP could inhibit the activation of p38 MAPK, JNK, NF-κB pathways and activate Nrf2 pathway. To further explore the possible transportation mechanism of LMP with human serum albumin (HSA), ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy were used to evaluate the interaction between LMP and HSA. The results showed that LMP-HSA complex was formed, which would be helpful for explaining the transportation mechanism in vivo. These results suggested that LMP might be a new therapeutic candidate to alleviate the high glucose toxicity.