O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory post-translational modification of nucleo-cytoplasmic proteins which consists in the attachment of N-acetylglucosamine to serine or threonine residues of a protein. This glycosylation is an ubiquitous post-translational modification which probably plays important roles in many aspects of protein function. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway and energetic metabolism, and contractile proteins were O-GlcNAc modified. O-GlcNAc has been recently demonstrated to play a role in modulating cellular function in response to nutrition and also in stress conditions. Therefore, we have investigated here the implication of the glycosylation/deglycosylation process in the development of atrophy in rat skeletal muscle after hindlimb unloading (HU). The high O-GlcNAc level found in control soleus (when compared to control EDL) becomes lower in atrophied soleus. On the opposite, the low rate of O-GlcNAc in control EDL reaches higher levels in EDL, not atrophied after HU. These variations in O-GlcNAc level are correlated with a variation of the O-GlcNAc process enzyme activities, and could be associated with a differential expression of heat shock proteins. Our results suggest that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.