O-GlcNAcylation is a post-translational modification of a protein that plays an important role in cancer pathogenesis and chemoresistance mechanisms. Osteosarcoma is the most common malignant bone tumor that occurs in children and young adults. If the cancer is diagnosed at the localized stage, the 5-year survival rate is > 65%; however, the rate decreases in metastatic cancer and poor responder chemotherapy. The genomic background is complex, and the implications of O-GlcNAc remain unclear in osteosarcoma. Aberrant O-GlcNAc was demonstrated in osteosarcoma using immunohistochemistry of O-GlcNAcylated modified proteins, O-GlcNAc transferase, and O-GlcNAcase to assess preoperative and postoperative chemotherapeutic specimens derived from the first 20 diagnosed osteosarcoma patients. The specimens were fixed in formalin and paraffin-embedded bone tissue sections. Differentiation expression levels of the O-GlcNAcylated proteins, O-GlcNAc transferase, and O-GlcNAcase between preoperative and postoperative chemotherapeutic specimens were analyzed using the Wilcoxon matched-pairs signed-rank test. Results presented significantly lower immunoreactivity scores of O-GlcNAcylated proteins (p=0.0083), while a lower O-GlcNAc transferase expression (p=0.2634) was observed in post-chemotherapeutic osteosarcoma specimens. In contrast, O-GlcNAcase expression showed higher levels in post-chemotherapeutic osteosarcoma specimens (p=0.7680). The findings indicated that O-GlcNAc was possibly related to tumorigenesis and may be useful for describing chemoresistance mechanisms of osteosarcoma.