The increasing use of lithium (Li) in emerging technologies has prompted concerns about its effects on living microorganisms. To enhance our understanding of the bacterial cytotoxicity of Li, we conducted a deletomic analysis using the bacterial model Escherichia coli. A screen of 3,985 knockout mutants under Li stress highlighted 27 Li-sensitive and 15 Li-resistant mutants. The synthesis of peptidoglycan and the capsule, along with the secretion of colanic acid, contributed to resistance to Li. Ribosomes and the stringent response also seem to play a role in mitigating Li cytotoxicity. A cross-metal comparison revealed that the Li-sensitive phenotype of the mutants was shared with Ca, whereas the resistant phenotype was shared with Mg, Na and K. Moreover, this allowed the identification of ΔacrA as a Li sensitivity-specific mutant. AcrA is a subunit of the AcrAB-TolC efflux pump, which is responsible for the efflux of various xenobiotics. We demonstrate that ΔacrB-ΔtolC accumulates approximately 1.5 times more Li than the WT, indicating that this pump could also facilitate the efflux of Li. This study offers a more comprehensive insight into the mechanisms involved in the Li response in E. coli.