We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous protein modification in eukaryotes, suppresses thermal inactivation of Sp1 transcription factor. 6-Diazo-5-oxonorleucine treatment or O-GlcNAcase overexpression, which reduced O-GlcNAc levels on Sp1, deteriorated thermal stability of Sp1 and O-GlcNAc modified molecules of Sp1 resist thermal aggregation in vitro. We also showed that heat-induced elevation of heat shock protein 70 was facilitated by Sp1 but blunted under low O-GlcNAc levels, suggesting that O-GlcNAc might upregulate the expression of heat shock protein 70 through thermoprotection of Sp1, which eventually enhanced cellular thermotolerance.