Sarcolipin (SLN) is a small protein,,that regulates the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in cardiac/skeletal muscles. Although it is reported that the Thr⁵ residue of human SLN (hSLN) is the key amino acid which modulates SLN function via its phosphorylation, it is not clear whether SLN is modified with O-linked b-N-acetylglucosamine (O-GlcNAcylation). Here, we found that mouse SLN (mSLN) was O-GlcNAcylated at Ser⁴ and Thr⁵ residues in mouse heart homogenates, by using an enzymatic labeling and a chemical detection system of O-GlcNAc. We also performed co-IP experiments using an O-GlaNAcase inhibitor in HEK293 cells where FLAG-tagged single Ala-substituted mutants to Ser⁴ /Thr⁵ residues of SLN (S4A/T5A) was expressed. As a result, the O-GlcNAcylation of SLN-WT was significantly increased with the O-GlaNAcase inhibitor, whereas no O-GlcNAcylation was detected in S4A/T5A both with or without the O-GlaNAcase inhibitor. Furthermore, SERCA activity was decreased with treatment of the O-GlaNAcase inhibitor in SLN-WT expressing HEK293 cells. These data indicate that the inhibition of SERCA activity by SLN is regulated not only via phosphorylation, but also via O-GlcNAcylation.