Human neutrophil elastase (HNE) has been shown to be involved on death of different cell types, including epithelial lung cells, which is related to several pulmonary diseases. Since HNE activity may be influenced by extracellular matrix (ECM) molecules such as glycosaminoglycans (GAGs), and fibroblasts are the most common ECM-producing cells of lung connective tissue, the aim of this work was to verify if HNE can induce fibroblast death and to study the enzyme modulation by GAGs. HNE-like activity was mimicked by using human neutrophils conditioned medium (NCM). Heparan sulfate and chondroitin 6-sulfate reduce the enzyme activity and modify its secondary structure. NCM reduced cell viability, and this effect was higher in the presence of those GAGs. NCM also increased DNA fragmentation, suggesting the occurrence of apoptosis, but without influence of GAGs. These results can contribute to the understanding of HNE modulation in physio- and pathological processes where this enzyme is involved.