Although dietary flavonoid quercetin alleviates diabetes-associated cognitive decline in rodents, the mechanisms are not clearly clarified. This study was designed to investigate whether quercetin showed neuroprotection on central neurons against chronic high glucose through the enhancement of Nrf2/ARE/glyoxalase 1 (Glo-1) pathway. SH-SY5Y cells were divided into 8 groups: normal glucose, high glucose (HG), osmotic pressure control, solvent control, HG plus low, middle, high concentrations of quercetin, or Nrf2 activator (sulforaphane). After treatment for 72 h, the associated parameters were measured. We found quercetin and sulforaphane increased cell viability, and enhanced Glo-1 functions (Glo-1 activity, the reduced glutathione and advanced glycation end-products levels) as well as Glo-1 protein and mRNA levels in SH-SY5Y cells cultured with HG. Meanwhile, quercetin and sulforaphane activated Nrf2/ARE pathway, reflected by the raised Nrf2 and p-Nrf2 levels, and the elevated protein and mRNA levels of γ-glutamycysteine synthase (γ-GCS), a known target gene of Nrf2/ARE signaling. Moreover, Nrf2/ARE pathway was activated after pretreatment with a PKC activator, p38 MAPK inhibitor, or GSK-3β inhibitor under the condition of HG, and quercetin addition further strengthened this pathway; however, PKC inhibition or GSK-3β activation pretreatment reversed the effects of quercetin on the protein expression of γ-GCS in the HG condition. In summary, quercetin exerts the neuroprotection by enhancing Glo-1 functions in central neurons under chronic HG condition, which may be mediated by activation of Nrf2/ARE pathway; furthermore, the increased Nrf2 phosphorylation mediated by PKC activation and/or GSK-3β inhibition may involve in the activation of Nrf2/ARE pathway.