Ewing Sarcoma is a bone sarcoma affecting most patients (~80%) before 18 years old and accounts for 2% of all childhood cancers. The most common cause of the disease is a chromosomal translocation of the Ewing sarcoma protein (EWS) from exons 1 – 6 to the gene encoding Fli1, an Ets family transcription factor, resulting in the EWS-Fli1 fusion protein. The EWS component drives high expression of the of the fusion protein, while the Fli1 region contains the DNA binding domain, and serves to activate specific genes commonly involved in megakaryocyte differentiation. Heparanase, an endoglycosidase already shown to be upregulated in several carcinomas, has also been recently implicated in Ewing sarcoma, with patient samples showing overexpression of heparanase and correlating with a poor prognosis. This study investigates the relationship between EWS-Fli1 and heparanase gene regulation. Here, we show that heparanase expression levels correlate with expression levels of EWS-Fli1 in Ewing Sarcoma lines CHLA9, 10 and 258. Luciferase reporter assays show increased activity of the heparanase promoter with increasing levels of EWS-Fli1, and overexpression of Fli1 increases heparanase mRNA expression and protein levels. Furthermore, chromatin immunoprecipitation reveals that Fli1 can directly bind to the heparanase promoter. From these findings, we conclude that the EWS-Fli1 fusion protein is capable of upregulating heparanase in Ewing sarcoma. These findings further aid our understanding of the mechanism of regulation of heparanase in the context of tumour progression and metastasis.