Gene therapy with adenoviral (Ad) vectors is a promising new approach in the treatment of cancer. Strategies to restrict adenoviral-mediated transgene expression are important to avoid gene transfer into normal cells. Heparanase (HPR) is overexpressed in breast cancer but downregulated in differentiated normal tissue. Expression of the HPR gene was evaluated in breast cancer cells. Biodistribution and liver tropism was evaluated in a mouse model. HPR is highly expressed in breast cancer tissue. The HPR promoter retained its fidelity in an adenovirus context and was activated in breast cancer cells but showed low activity in normal breast cells and the murine liver. We conclude that the HPR pathway is a promising target for the development of breast cancer directed gene therapy strategies.