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.