Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.