O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies thousands of proteins. However, the roles and mechanisms of O-GlcNAcylation have been clarified in only a few proteins, and one of the main reasons for this is the lack of site-specific anti-O-GlcNAc antibodies. Recently, we found that SIRT1, which is an NAD+-dependent deacetylase, is O-GlcNAcylated at the serine 549 site (S549) and plays a cytoprotective role under stress. However, the mechanism underlying the roles of SIRT1 O-GlcNAcylation remains unclear. Here, we describe a site-specific antibody for SIRT1 O-GlcNAcylated at S549, named SIRT1-549-O. This antibody can be used for immunoprecipitation and western blotting assays, and it can be used to recognize the endogenous levels of both human and mouse SIRT1 O-GlcNAcylation. Therefore, this antibody not only provides an effective method to further understand the roles of SIRT1 O-GlcNAcylation but also makes it possible to discover the genetic and pharmacological factors that could regulate SIRT1 activity by modulating its O-GlcNAcylation.