Lactoferrin (Lf) is a multifunctional protein that has inhibitory activity against microorganisms. In this study, the effects of Lf on the growth of human parainfluenza virus type 2 (hPIV-2) in LLCMK₂ cells were investigated. Lf inhibited cell fusion and hemadsorption induced by hPIV-2. However, virus RNA synthesis was only slightly inhibited by Lf. In addition, indirect immunofluorescence study showed that virus protein syntheses were not completely inhibited by Lf. Using a recombinant, green fluorescence protein-expressing hPIV-2 (rghPIV-2), it was found that virus entry into cells were considerably inhibited by Lf, but cell-to-cell spread was not inhibited. The number of viruses produced by the cells were determined, and it was found that Lf reduced the number of released viruses to about 1/300 compared with that of positive control. Lf bound to cell surface within 10 min at early phase of infection, it assembled gradually, and many aggregates were observed at 120 min. These results indicated that Lf considerably inhibited virus adsorption to the surface of the cells by binding to the cell surface and prevented hPIV-2 infection.