Aflatoxins are a group of potent fungal metabolites produced by Aspergillus and commonly contaminate groundnuts and cereal grains. Aflatoxin B 1 (AFB 1 ), the most potent mycotoxin, has been classified as Group 1 human carcinogen because it can be metabolically activated by the cytochrome P450 (CYP450) in the liver to form AFB 1 -DNA adducts and induce gene mutations. Increasing evidence has shown the gut microbiota as a key mediator of AFB 1 toxicity through multiple interactive host-microbiota activities. To identify specific bacterial activity that modulates AFB 1 toxicity in Caenorhabditis (C.) elegans, we established a 3-way (microbe-worm-chemical) high-throughput screening system using C. elegans fed E. coli Keio collection on an integrated robotic platform, COPAS Biosort. We performed 2-step screenings using 3985 Keio mutants and identified 73 E. coli mutants that modulated C. elegans growth phenotype. Four genes (aceA, aceB, lpd, and pflB) involved in the pyruvate pathway were identified from the screening and confirmed to increase the sensitivity of all animals to AFB 1 . Taking together, our results indicated that disturbances in bacterial pyruvate metabolism might have a significant impact on AFB 1 toxicity in the host.