In this study, we demonstrated that OTX2 was transcriptionally and post-transcriptionally regulated by O-GlcNAcylation. We describe the modes of degradation of this essential homeobox protein involving both the proteasome and macroautophagy. While the proteasome remains the canonical degradation pathway, we demonstrated that OTX2 forms oligomers and/or aggregates that are resistant to proteasomal degradation and require macroautophagy. Furthermore, elevated cellular concentration of OTX2 promoted aggregation, OTX2 could either interact with the chaperonin CCT5 or be O-GlcNAcylated at the key residues (S1135/S136/T137) to prevent its oligomerization, suggesting a protective role of O-GlcNAcylation in OTX2 turnover.