Although calcium is the major regulator of excitation-contraction coupling, myofilament function can also be modulated through post-translational modifications. In particular, phosphorylation and O-GlcNAcylation are key modulators of calcium activation parameters. Among the regulatory proteins of skeletal muscle contraction, the myosin light chain 2 (MLC2) can undergo both types of post-translational modification. During aging or physical inactivity, the phosphorylation status of the slow isoform of MLC2 (sMLC2) does not correlate with calcium sensitivity, suggesting that the O-GlcNAcylation might modulate sMLC2 activity. To increase understanding of the contractile dysfunction associated with muscle atrophy, we studied the phosphorylation/O-GlcNAcylation interplay on the sMLC2. We demonstrate a two-fold decrease of O-GlcNAcylation level on sMLC2 in a rat model of skeletal muscle atrophy (hindlimb unloading), while phosphorylation increased. Both post-translational modifications were mutually exclusive. Their interplay reversed during reloading. The expression of enzymes involved in the phosphorylation and O-GlcNAcylation interplay on sMLC2 was modified on whole protein pattern as well as on myofilament, and was load-dependent. All enzymes were colocalized on the contractile apparatus. Finally, we describe a multienzymatic complex which might finely modulate the phosphorylation/dephosphorylation and O-GlcNAcylation/de-O-GlcNAcylation of sMLC2 that could be involved in the contractile dysfunction of atrophied muscle. Importantly, this complex was localized at the Z-disk, a nodal point of signalling in skeletal muscle.