Yersinia enterocolitica induces a broad range of gastrointestinal syndromes, including acute enteritis. We previously reported that the clinical isolate, Y. enterocolitica KU14, which lacks pYV, was still capable of causing clinical infection. The present study demonstrated that KU14 did not trigger the death of macrophages in vitro, unlike WA-314 (ATCC51871, which harbors the pYV virulence plasmid). However, the intracellular growth of KU14 in the macrophages was greater than that of WA-C (ATCC51872, a non-plasmid harboring the derivative pYV plasmid). Treatment with a cholesterol-binding drug (β-cyclodextrin) that affected lipid rafts resulted in a dramatic reduction in the intracellular growth of KU14. These data clearly indicate that the enhanced intracellular growth of KU14 is related to lipid raft-mediated infection.