Fibrosarcoma is an uncommon soft tissue tumor with a complex cell microenvironment, particularly rich in glycosaminoglycans/proteoglycans (GAGs/PGs). Chondroitin sulfate proteoglycans (CSPGs) participate in the modulation of various cellular functions, including adhesion and migration. The role of chondroitin sulfate (CS) chains on adhesion, chemotaxis and migration of poorly differentiated fibrosarcoma B6FS cell was studied, utilizing exogenous CS treatment and chondroitinase digestions as well as specific modulators of CS synthesis. Cleavage of cell-associated CS chains and specific inhibition of endogenous CS production severely impaired these fibrosarcoma cell functions. These results show that the reduction of endogenous CSPG expression as well as cleavage of the CS chain inhibited fibrosarcoma cell motility, migration and adhesion. Treatment with free CS chains enhanced cell chemotaxis and migration, whereas adhesion was inhibited. CS chains were found to upregulate cell motility through the MAPK pathway, specifically through JNK, whereas CS-induced migration was found to require tyrosine kinase dependent pathways. This study suggests a new role of CS on tumor cell adhesion, chemotaxis and migration.