Flavonoid quercetin and its derivative, methylquercetin, inhibit the replication of poliovirus in several cell lines. Here, we show that replication of poliovirus is inhibited by quercetin and that the extent of this inhibition depends on the intracellular content of pirin, a quercetinase. HeLa cells contain higher content of pirin protein than normal kidney human epithelial (NKE) or 293 cells do. Poliovirus replication in HeLa cells is significantly more resistant to quercetin than its replication in NKE and 293 cells. Overexpression of pirin reduced antiviral inhibitory effect of quercetin, while siRNA-induced suppression of pirin level made poliovirus replication more sensitive to the flavonoid. The results suggest that quercetinase activity of pirin determines the resistance of poliovirus infection to quercetin.