Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTI), a major global public health concern associated with significant morbidity and mortality. All pathogens must breach the innate immune system to cause disease; thus, an understanding of the interactions between this system and UPEC may lead to new therapeutic approaches for UTI. We recently showed that some UPEC strains rapidly kill human macrophages, likely as a host evasion mechanism. Using a random transposon mutagenesis library in the reference strain CFT073, we identified eight clones that were impaired in their ability to kill primary human macrophages. These hits were independently validated and sequenced to identify mutated genes, and independent knock-outs were generated to further validate the findings. This approach revealed that the hemolysin A (hlyA) toxin was essential for triggering both cell death and inflammasome-dependent IL-1β release in human macrophages. The poorly characterized cof gene was also identified as a novel hemolysin regulator; a CFT073∆cof was compromised for hlyA secretion, and was impaired in its capacity to trigger human macrophage cell death. In surveying isolates of the globally disseminated E. coli sequence type 131 (ST131) clone, we found that only hlyA+vestrains caused human macrophage cell death. Cell death correlated with inflammasome activation, but was independent of the NLRP3 inflammasome. In contrast, IL-1β release was completely NLRP3-dependent. hlyA was identified as the virulence factor responsible for ST131-induced cell death and IL-1β release in human macrophages. Nonetheless, there was considerable variability between hlyA+ve ST131 strains in the magnitude of these responses, suggesting the involvement of additional factors. Overall, this study highlights that hlyA enables UPEC-initiated inflammasome activation and cell death in human macrophages, and that an NLRP3-independent cell death pathway exists in these cells. We are currently investigating the role of UPEC-triggered macrophage cell death in host evasion and virulence.