Enteric bacteria possess multiple fimbriae, many of which play critical roles in attachment to epithelial cell surfaces. SEF14 fimbriae are only found in Salmonella enterica serovar Enteritidis (S. enteritidis) and closely related serovars, suggesting that SEF14 fimbriae may affect serovar-specific virulence traits. Despite evidence that SEF14 fimbriae are expressed by S. enteritidis in vivo, previous studies showed that SEF14 fimbriae do not mediate adhesion to the intestinal epithelium. Therefore, we tested whether SEF14 fimbriae are required for virulence at a stage in infection after the bacteria have passed the intestinal barrier. Polar mutations that disrupt the entire sef operon decreased virulence in mice more than 1,000-fold. Nonpolar mutations that disrupted sefA (encoding the major structural subunit) did not affect virulence, but mutations that disrupted sefD (encoding the putative adhesion subunit) resulted in a severe virulence defect. The results indicate that the putative SEF14 adhesion subunit is specifically required for a stage of the infection subsequent to transit across the intestinal barrier. Therefore, we tested whether SefD is required for uptake or survival in macrophages. The majority of wild-type bacteria were detected inside macrophages soon after i.p. infection, but the sefD mutants were not readily internalized by peritoneal macrophages. These results indicate that the potential SEF14 adhesion subunit is essential for efficient uptake or survival of S. enteritidis in macrophages. This report describes a role of fimbriae in intracellular infection, and indicates that fimbriae may be required for systemic infections at stages beyond the initial colonization of host epithelial surfaces.