Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.