The present study aimed to investigate the protective effect of purified polysaccharides from Tremella fuciformis against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The aqueous extract of Tremella fuciformis was purified using a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column, respectively. A fraction termed TL04 with a 2,033 kDa molecular weight was obtained. The backbone of TL04 is composed of (1→2)‑ and (1→4)-linked-mannose and (1→3)-linked-glucans. Results revealed that TL04 treatment improved cell viability and suppressed reactive oxygen species accumulation, lactose dehydrogenase release and caspase-3 activity, and ameliorated mitochondrial abnormal alteration caused by glutamate. TL04 pretreatment enhanced the level of B‑cell lymphoma 2 (Bcl‑2), and suppressed Bax expression and cytochrome c (Cyto C) release in glutamate-treated cells. Exposure to glutamate strongly increased the activity of caspase‑8, caspase‑9 and caspase‑3, which were significantly reversed by TL04 pretreatment. The presence of Ac-DEVD-CHO (a caspase-3 inhibitor) markedly enhanced the potency of TL04 in improving the viability of glutamate-exposed DPC12 cells. Collectively, the results demonstrated that the purified polysaccharides separated from Tremella fuciformis (TL04) possess a neuroprotective effect against glutamate-induced DPC12 cell damage predominantly through the caspase-dependent mitochondrial pathway. The present study provides an experimental foundation supporting purified TL04 as a potential therapeutic agent for neurodegenerative diseases.