Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Here, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal profiling of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. Substantial shifts in gut microbial community structure during oral fluoropyrimidine treatment across multiple patient cohorts, in mouse small and large intestinal contents, and in patient-derived ex vivo communities were revealed by 16