Addition of methylated β-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 μg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO2. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 μg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 μg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.