Curli fibers are adhesive surface fibers expressed by Escherichia coli and Salmonella enterica that bind several host extracellular matrix and contact phase proteins and were assumed to have a role in pathogenesis. The results presented here suggest that one such role is internalization into host cells. An E. coli K-12 strain transformed with a low-copy vector containing the gene cluster encoding curli fibers (csg operon) was internalized by several lines of eukaryotic cells. The internalization could be correlated with a high level of curli fiber expression and was abolished by disruption of the csg operon. The ability to be internalized by eukaryotic cells could be conferred even by the curli fiber gene cluster of a noninvasive K-12 strain, but the homologous csg cluster from a virulent septicemic E. coli isolate mediated a higher level of internalization. The finding that curli fibers promote bacterial internalization indicates a new role for curli fibers in pathogenesis.