A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium HPLM). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis—an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility. Display omitted •Systematic development of human plasma-like medium (HPLM)•Culture in HPLM has widespread effects on cellular metabolism•Culture in HPLM reveals uric acid as an endogenous inhibitor of UMPS•Uric acid can influence cellular sensitivity to 5-fluorouracil Mimicking the metabolite composition of human plasma in culture extensively alters the metabolic landscape of cells and highlights the potential to uncover new metabolite-drug interactions.