-GlcNAcylation is a post-translational modification of proteins that controls a variety of cellular processes, is chronically elevated in diabetes mellitus, and may contribute to the progression of diabetic complications, including diabetic nephropathy. Our previous work showed that increases in the -GlcNAcylation of cellular proteins impair the homeostatic reaction of the regulatory volume decrease (RVD) after cell swelling by an unknown mechanism. The activation of the swelling-induced chloride current IClswell is a key step in RVD, and ICln, a ubiquitous protein involved in the activation of IClswell, is -GlcNAcylated. Here, we show that experimentally increased -GlcNAcylation of cellular proteins inhibited the endogenous as well as the ICln-induced IClswell current and prevented RVD in a human renal cell line, while decreases in -GlcNAcylation augmented the current magnitude. In parallel, increases or decreases in -GlcNAcylation, respectively, weakened or stabilized the binding of ICln to the intracellular domain of α-integrin, a process that is essential for the activation of IClswell. Mutation of the putative YinOYang site at Ser67 rendered the ICln-induced IClswell current unresponsive to -GlcNAc variations, and the ICln interaction with α-integrin insensitive to -GlcNAcylation. In addition, exposure of cells to a hypotonic solution reduced the -GlcNAcylation of cellular proteins. Together, these findings show that -GlcNAcylation affects RVD by influencing IClswell and further indicate that hypotonicity may activate IClswell by reducing the -GlcNAcylation of ICln at Ser67, therefore permitting its binding to α-integrin. We propose that disturbances in the regulation of cellular volume may contribute to disease in settings of chronically elevated -GlcNAcylation, including diabetic nephropathy.