Melanoma is a global health issue, especially in Australia, where it has the highest incidence in the world. Despite advances in treatment and earlier detection, melanoma is still the most common metastatic skin cancer. Although a large possibility of treatment is available for this cancer, e.g. chemotherapy and immunotherapy, many patients still display refractory and resistant melanoma. Hence, the investigation of new compounds to treat malignant melanoma is necessary. Heparin is a promising candidate since it is already administrated in cancer patients to avoid venous thromboembolism. It was previously shown that heparin could inhibit the expression of proto-oncogenes and reduce the activity of heparanases and metalloproteinases in some tumour cells. The anticoagulation activity is the most studied aspect of heparin, because it can inhibit the interactions between tumour cells and platelets, impairing the metastases process and making cancer cells detectable for the immune system. The aim of this work was to evaluate the effects of heparin and its de-2-O-sulfated (D2OS) derivative on B16F10 murine melanoma cells. First, cells were exposed in vitro to different concentrations in a time-point kinetics to heparin and D2OS. Although both compounds did not show potential cytotoxic effect or changes on cellular proliferation, it modulated the invasion profile of B16F10 cells and their clonogenic capacity. We next measured the effects on the invasion capacity, where pre-treated cells were seeded in transwells inserts coated with matrigel and cultured for 72 h, and then the clonogenic assay cells were seeded at algimatrix 3D culture system and maintained for 144 h. Interestingly, the treatment with the D2OS heparin derivative reduced the invasion of melanoma cells and the size of colonies. Subsequently in vivo treatment of subcutaneously B16F10-bearing mice with heparin or D2OS (10 mg/kg) daily for 10 days also resulted in diminished tumour growth over time. These results suggest a novel application of heparins, and it’s strategically de-sulfation can be used in appropriate therapeutic interventions to promote anti-tumour responses.