Acute pyelonephritis, frequently caused by Escherichia coli, is a substantial health problem. Plasminogen activator inhibitor type-1 (PAI-1) not only inhibits plasminogen activation but is also involved in cell migration. To determine if it has a role in host defense, we induced pyelonephritis in PAI-1 gene knockout and wild-type mice by intravesical inoculation with uropathogenic E. coli 1677. Bacterial growth was determined on blood agar plates in portions of the kidneys homogenized in sterile saline. Kidney levels of PAI-1 were increased in infected compared to control mice, suggesting a physiological role for PAI-1 during pyelonephritis. The knockout mice had significantly more bacterial outgrowth in kidney homogenates compared to the wild-type mice. Strikingly, higher colony-forming units were accompanied by increased levels of the cytokines TNF-α, IL-1β, and IL-6 in the kidneys of knockout mice, but levels of the chemokines KC and MIP-2 were not different. Remarkably, plasma levels of KC were higher, but renal neutrophil influx was significantly lower, in the knockout than in the wild-type mice. Our study shows that PAI-1 is critically involved in host defense against E. coli-induced acute pyelonephritis, in part, by modulating neutrophil influx.