Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca²⁺ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because <italic>O</italic>-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 <italic>O</italic>-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 <italic>O</italic>-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 wild-type (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible <italic>O</italic>-GlcNAcylation sites of STIM1 to determine whether the sites were <italic>O</italic>-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were <italic>O</italic>-GlcNAcylated and that Thr626 was <italic>O</italic>-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621. Treatment with the <italic>O</italic>-GlcNAcase inhibitor Thiamet G or <italic>O</italic>-GlcNAc transferase (OGT) transfection, which increases <italic>O</italic>-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases <italic>O</italic>-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in <italic>O</italic>-GlcNAcylation was attributable to reduced phosphorylation at Ser621. These data suggest that both decrease in <italic>O</italic>-GlcNAcylation at Thr626 and increase in<italic> O</italic>-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 <italic>O</italic>-GlcNAcylation could provide a promising treatment option for the related diseases such as neurodegenerative diseases.