In an era when cancer glycobiology research is exponentially growing, we are witnessing a progressive translation of the major scientific findings to the clinical practice with the overarching aim of improving cancer patients’ management. Many mechanistic cell biology studies have demonstrated that heparan sulfate (HS) glycosaminoglycans are key molecules responsible for several molecular and biochemical processes, impacting extracellular matrix properties and cellular functions. HS can interact with a myriad of different ligands, and therefore, hold a pleiotropic role in regulating the activity of important cellular receptors and downstream signalling pathways. The aberrant expression of HS glycan chains in tumours determines main malignant features, such as cancer cell proliferation, angiogenesis, invasion and metastasis. In this review, we devote particular attention to HS biological activities, its expression profile and modulation in cancer. Moreover, we highlight HS clinical potential to improve both diagnosis and prognosis of cancer, either as HS-based biomarkers or as therapeutic targets.