The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.