Single N-acetylglucosamine residues attached by O-linkage to serine or threonine (O-GlcNAc) are an abundant, dynamic and inducible post-translational modification of cytoplasmic and nuclear proteins. This study analyzes the activity of the enzyme involved in the removal of these sugar residues, i.e. beta-N-acetylglucosaminidase (O-GlcNAcase) as well as the level of O-GlcNAc in benign and malignant thyroid lesions. Our results demonstrate increased activity of the enzyme in thyroid cancers in comparison to non-neoplastic lesions and adenomas. O-GlcNAc-modified proteins in thyroid cells have a predominantly nuclear distribution and are more abundant in non-neoplastic lesions than in tumors. Understanding the aberrant O-GlcNAc metabolism in thyroid cancer cells may be helpful for developing new diagnostic or treatment methods.