Enterococcus faecalis is a gram-positive bacterium that can cause a variety of nosocomial infections of which urinary tract infections are the most common. These infections can be exceptionally difficult to treat because of drug resistance of many E. faecalis isolates. Despite their troublesome nature, little is known about the host or bacterial factors necessary for E. faecalis to cause disease in the urinary tract. Using a mouse model of urinary tract infection, we have shown that E. faecalis is capable of persisting in the kidneys of mice for at least 2 weeks. In contrast, bacterial titers from the bladders of the same mice were inconsistent and tended to be much lower than those recovered from the kidney. This preference for the kidney over the bladder is also observed in other clinical E. faecalis strains. Histologic examination of bladder and kidney tissues demonstrated that E. faecalis induced an inflammatory response in the kidney but not in the bladder. This inflammatory response was TLR2 independent and did not induce inflammatory markers typically associated with uropathogenic Escherichia coli. Using a competition assay, we demonstrated that a pyelonephritis clinical isolate had a growth advantage over a laboratory strain of E. faecalis in the kidneys but not in the bladders of mice. Taken together, these results demonstrate that E. faecalis has tropism for the kidneys in the urinary tracts of mice and that this system can be used to study factors involved in the pathogenesis of urinary tract infections.