We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC–ESI–MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo. Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.