Objective To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.