4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibitingHAsynthesis. In particular,4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent4-MU:4-MUGratios in serum, liver, and pancreas, indicating that4-MUand4-MUGreachanequilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to4-MUin serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, wefound that4-MUG(a nonfluorescent molecule) undergoes conversion into4-MU(a fluorescent molecule)andthat4-MUis extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibitsHAsynthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition. [ABSTRACT FROM AUTHOR]