ABSTRACT A comprehensive Tn phoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1 ⁺ ) Pgm ⁻ (pigmentation negative)/, KIM6(pCD1 ⁻ ) Pgm ⁺ , and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro , infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δ ilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain ( P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δ ilp , mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system ( in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δ ilp had a 55-fold increase in the 50% lethal dose ([LD ₅₀ ] 1.64 × 10 ⁴ CFU) compared to the parental wild-type strain LD ₅₀ (2.98 × 10 ² CFU). These findings identified Ilp as a novel virulence factor of Y. pestis .