Reactive carbonyl compounds contribute to aging, Alzheimer's disease (AD) and other neurodegenerative diseases. Among these compounds, methylglyoxal (MG) can yield advanced glycation end products (AGEs), which are crucial in AD pathogenesis. However, the molecular and biochemical mechanisms of MG neurotoxicity are not completely understood. In the present study, SH‐SY5Y cells were treated with MG to induce cell death. 2‐D Fluorescence Difference Gel Electrophoresis and matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry were employed to determine the changes in protein levels in these cells compared with vehicle‐treated cells. Proteomics analysis revealed that 49 proteins were differentially expressed in MG‐treated SH‐SY5Y cells, of which 16 were upregulated and 33 were downregulated. Among them, eight proteins were identified unambiguously. The significant changes in protein levels of actin, immunoglobulin lambda light chain and protein phosphatase 2 were noteworthy given their functional roles in AD pathogenesis. Taken together, our results suggest that multiple pathways are potentially involved in MG‐induced neuron death. Copyright © 2010 John Wiley & Sons, Ltd.