Diabetes mellitus is one of the risk factors of neurological disorders such as Alzheimer’s disease (AD). Accumulation of advanced glycation end products (AGEs) including glucose-derived AGEs, glyceraldehyde (GA)-derived AGEs (toxic AGEs, TAGEs) in the brain with aging was proposed to be involved in pathogenesis of AD. We previously confirmed that TAGEs are strongly neurotoxic. Furthermore, in AD patient brains, TAGE but not glucose-AGEs were mainly detected in the cytosol and axons of neurons in the hippocampus, but not in senile plaques. However, molecular targets of GA remain unclear. In this study, we investigated the target protein for TAGE by performing two-dimensional immunoblot analysis with anti-TAGE antibody and mass spectrometry and identified β-tubulin as one of the targets using SH-SY5Y cell lines. Glucose did not affect to β-tubulin physiological functions and did not inhibit neurite outgrowth. However, GA induced TAGE-β-tubulin formation and abnormal aggregation of β-tubulin, and inhibited neurite outgrowth in SH-SY5Y cells. Understanding the mechanism of TAGE-β-tubulin formation by GA and its role in neurodegeneration may aid in the development of novel therapeutics and neuroprotection strategies.