Uropathogenic E. coli (UPEC) is the main etiological agent of urinary tract infection (UTI), one of the most prevalent community acquired and nosocomial infections in countries with high socioeconomic status. The recent emergence of multidrug-resistant UPEC strains, as well as the prevalence of chronic or reoccurring infections, necessitates a deeper understanding of the pathophysiological mechanisms of UTI. Although the innate immune system has been shown to to be central for controlling UTI, there is surprisingly little known of the interactions between UPEC and macrophages, one of the key cell types orchestrating innate immunity. Here we show that some of the most widely studied UPEC strains (CFT073 and UTI89) trigger activation of the NLRP3 inflammasome, a multi-protein complex sensing cellular stress and microbial products, in primary human macrophages. This leads to IL-1beta secretion and rapid pyroptotic cell death. In contrast, other UPEC strains, including clinical isolates of the emerging ST131 lineage, did not elicit these responses. Whereas CFT073 also triggered NLRP3-dependent responses in primary mouse macrophages, UTI89 did not. Since UTI89 has been widely employed in mouse models to study mechanisms of UTI pathology, our findings highlight the need for careful examination of species-differences in innate immune recognition pathways. Moreover, our findings suggest that distinct clinical isolates of UPEC can have fundamentally different interactions with the innate immune system, consistent with the broad genetic diversity across UPEC strains. Our current studies are examining the mechanistic basis for species differences in UPEC detection, as well as the bacterial factor(s) responsible for NLRP3-dependent inflammasome responses in human macrophages.