O -Linked β- N -acetylglucosamine-modification ( O -GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O -GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O -(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N -phenyl carbamate (PUGNAc), an inhibitor of O -GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O -GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O -GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O -GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O -GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O -GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O -GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O -GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O -GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper- O -GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.