3T3-L1 adipocytes develop insulin resistant glucose transport upon preincubation with high (25 mM) glucose, provided insulin (0.6 nM) is included; Akt activation is impaired; high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s), which may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis: 1. Overexpression of O-GlcNAcase which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase expressing adenovirus (or empty virus) 5 days before submitting them to protocols, which elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay and by marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. 2. The expression of O-GlcNAc transferase (OGT) was markedly reduced by transfection of OGT siRNA, resulting in ~90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Non-targeting siRNA had no effect. Preincubation in high glucose with low dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.