The inhibitory effect of a polysaccharide from kefir on human rotavirus infection in MA-104 cells was investigated. The extracted polysaccharide was separated as fraction I in unbound materials and as fractions II, III, and IV in bound materials. Adding polysaccharide fractions II (4.8 mg/mL), III (5.3 mg/mL), and IV (1.4 mg/mL) inhibited the infection of MA-104 cells by human rotavirus. The 50% inhibitory concentrations (IC 50) were 0.075, 0.083, and 0.022 mg/mL, respectively. Based on these results, the kefir polysaccharide has anti-rotavirus activity. In conclusion, a polysaccharide from kefir had more than a 97% inhibition effect against human rotavirus infection in MA-104 cells.