Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant stroma, proteoglycans provide a reservoir of potential new targets for anticancer therapies, because they can serve to convey toxic payloads in the close proximity of cancer cells and subsequently destroy them. In this context, versican, a proteoglycan largely overexpressed in several solid cancers, bears the potential to be such an ideal target. As 4 main versican isoforms have been characterized, we sought to determine which isoform could represent the best target in human breast cancer. We used a series of 10 primary breast cancer lesions that were characterized as overexpressing the versican protein, when compared with matched normal breast tissues, using shotgun mass spectrometry and immunohistochemistry experiments. Quantitative polymerase chain reaction and western‐blotting experiments were used to evaluate versican isoform expression in breast cancer/normal tissue pairs for which ARN quality was excellent. All known isoforms were significantly overexpressed in the malignant lesions, both at the mRNA and at the protein levels. In the course of this study, we also identified and cloned a new alternatively spliced versican isoform, referred to as V4, which was also found to be upregulated in human breast cancer. This study provides for the first time a comprehensive mRNA and protein analysis of versican isoforms expression in human breast tissues, and offers insights into which therapeutic strategy would be best suited to target versican in human breast cancer lesions.