O-linked b-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of the stromal interaction molecule 1 (STIM1) is known to impair store-operated Ca²⁺ entry (SOCE). In cardiomyocytes, STIM1 interaction with phospholamban (PLN), an inhibitor of Ca²⁺-ATPase (SERCA2a), is reported to regulate cardiac function. However, the molecular mechanisms by which STIM1 O-GlcNAcylation affects SOCE and PLN activity are still unclear. Here, we found that increased STIM1 O-GlcNAcylation with an O-GlcNAcase inhibitor reduced its interaction with PLN in HEK293 cells co-expressing myc-tagged STIM1 and GFP-tagged PLN-WT or S16A (mutant of O-GlcNAcylation site). The STIM1 interaction with PLN was significantly decreased in heart tissues of O-GlcNAc transferase-transgenic mice, whereas the PLN interaction with SERCA2a was significantly increased in that of STIM1-knockout mice compared to WT mice. These data suggest that STIM1 O-GlcNAcylation regulates its interaction with PLN, which may possibly regulate SERCA activity. Next, to clarify the molecular mechanisms by which STIM1 O-GlcNAcylation affects SOCE, we established the STIM1 knockout HEK293 cells by CRISPR/Cas9 system, and then, transfected STIM1 mutants lacking each O-GlcNAcylation site to the cells. It is being elucidated.