DescriptionBoth mammalian and bacterial cells respond to environmental cues and then elicit various physiological responses. Mechanical cues, such as surface attachment, have been noted as a critical part of bacterial infection through interaction with mechanosensors and the actin cytoskeleton. Urinary tract infections are a sufficient model for studying attachment due to the mechanism of bacterial infection. The cytoskeleton mediates various cell processes that are involved with infection, such as endocytosis and vesicle trafficking. The disruption of the host actin cytoskeleton leads to increased uropathogenic E. coli infection (UPEC). Since substrate stiffness mediates changes in host cell physiology through cytoskeletal interaction, we explored the UPEC infection process on polyacrylamide gels of differing stiffnesses. We found that UPEC escapes endocytic vesicles and replicates within the cytoplasm on soft gels, yet are trapped within lysosomal-associated membrane protein (LAMP-1) vesicles on stiff surfaces. Cytoplasmic replication is consistent with previous in vivo studies. Increased infection was also found on soft gels when compared with stiff substrates. These findings may provide a more accurate cell culturing model for future UPEC infection studies.