Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.