O‐GlcNAcylation on serine/threonine is a post‐translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O‐GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O‐GlcNAcylation in the context of pancreatic β‐cell glucotoxicity. FoxO1 was O‐GlcNAcylated in INS‐1 832/13 β cells and isolated rat pancreatic islets. O‐GlcNAcylation of FoxO1 resulted in a 2‐fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3‐fold increase in the expression of the insulin‐like growth factor‐binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin‐like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O‐GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS‐1 832/13 cells from high‐glucose‐ or O‐glcNAcylation‐induced cell death. In contrast, FoxO1 down‐regulation by siRNA led to 30 to 60% protection of INS‐1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O‐GlcNAcylation inhibits Akt through an IGFBP1‐mediated autocrine pathway, the deleterious effects of FoxO1 O‐GlcNAcylation on cell survival appeared to be independent of this pathway.—Fardini, Y., Masson, E., Boudah, O., Ben Jouira, R., Cosson, C., Pierre‐Eugene, C., Kuo, M.‐S., Issad, T. O‐GlcNAcylation of FoxO1 in pancreatic β cells promotes Akt inhibition through an IGFBP1‐mediated autocrine mechanism. FASEB J. 28, 1010–1021 (2014). www.fasebj.org