Infant formula supplemented with hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) was evaluated in vitro against HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274. Specific antihuman rotavirus antibody powder (Rotamix IgY) was prepared. The effectiveness of Rotamix IgY alone and as a pre-mixed solution with infant formula was evaluated for neutralizing rotavirus infectivity in MA104 cells. The test infant formula cross-reacted strongly against different human rotavirus strains with titers of 80-320 using a 50% fluorescent focus (FF) inhibition test. Both rotamix IgY alone and in a pre-mixed solution with infant formula showed multi-serotypic cross neutralization activities against the major rotavirus global serotypes G1, G2, G3, and G4 alone and with other human and animal-strains in vitro. Cell-rotavirus adhesion and cell damage arising from rotavirus infection were significantly inhibited in a dose dependent manner, compared to control IgY supplemented infant formula.