Objective Enteric glial cells (EGCs) can activate multiple pathways to inhibit the deleterious effects of acute and chronic insults. Our aim was to test the effect of EGCs on hyperglycemia-induced neuron damage and its underlying intracellular mechanisms. Methods A coculture model composed of EGCs and neuroblastoma cells (SH-SY5Y) was established to examine glial-mediated neuroprotection under high glucose conditions. The cell counting assay kit CCK-8 was used to measure cell viability. Flow cytometry was used to measure the induction of reactive oxygen species (ROS), change of mitochondrial membrane potential (MMP). cell cycle distribution, and apoptosis. The expressions of cyclin D1, cyclin E2, Bax, cleaved caspase-3, AKT, p-AKT, GSK-3 beta, and p-GSK-3 beta were tested using western blot. Results Exposure to high glucose (>= 35 mM) reduced the viability of SH-SY5Y cells in a concentration- and time-dependent manner. Meanwhile, enhanced ROS generation and decrease of MMP were observed in SH-SY5Y cells when treated with high glucose. Furthermore, high glucose also caused SH-SY5Y cells arrest in G2 phase and apoptosis, accompanied by decreasing cyclin D1 and E2. and upregulating Bax and cleaved caspase-3. Coculture EGC lines or EGC-conditioned medium with SH-SY5Y prevented the neurotoxic effects. The p-AKT/AKT and p-GSK-3 beta/GSK-3 beta) ratios were dramatically decreased in S H-SY5Y cells after high glucose incubation, which was restored after coculture with EGCs. Conclusions EGCs can protect neurons from hyperglycemia-induced injury by activating the Akt/GSK-3 beta; pathway. Copyright (C) 2021 Wolters Kluwer Health, Inc. All rights reserved.