Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections, of increasing concern due to their prevalence and the frequency of antibiotic resistance. Recent research suggests that zinc may be harnessed directly by innate immune cells for the destruction of engulfed microbes. However, the molecular mechanisms underpinning zinc toxicity are not well understood, and no studies have investigated whether this response is deployed against UPEC. Here, we report the development of novel zinc stress reporter strains in pathogenic and non-pathogenic E. coli in which the promotor of zntA, which encodes a zinc exporter selectively induced by zinc, drives mCherry expression. Utilising these strains, we monitored the antimicrobial zinc toxicity response at a single-cell level and found that primary human macrophages subject a non-pathogenic E. coli K-12 strain (MG1655) to zinc poisoning. In contrast, EC958, a representative of the multidrug resistant and globally-disseminated ST131 lineage of UPEC, effectively evades the macrophage zinc toxicity response. We validated these findings through analysis of a panel of zinc-sensitive mutants, which we identified through a genetic screen employing transposon-directed insertion site sequencing. Evasion of the zinc toxicity response by EC958 was also apparent in a mouse intraperitoneal infection model, whereas non-pathogenic E. coli was subjected to zinc toxicity within cells of the peritoneal cavity. However, reduced systemic dissemination of a zntA mutant strain revealed that UPEC employs both zinc evasion and resistance strategies for full virulence. In investigating host cell mechanisms responsible for zinc toxicity, we identified that the zinc exporter SLC30A1 is Toll-like Receptor-inducible in human macrophages. Utilising a doxycycline-inducible lentiviral overexpression system in the macrophage-like cell line THP-1, we report that SLC30A1 localises to both the plasma membrane and intracellular zinc-containing vesicles of macrophages. Overexpression of this transporter enhanced zinc stress and reduced intramacrophage survival of E. coli. Collectively, our studies reveal new insights into the deployment of zinc toxicity by macrophages, as well as evasion mechanisms used by a globally-disseminated bacterial pathogen against this pathway.