Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid alpha2,3 and alpha2,6 linkage preferences. The branched alpha2,3-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,4-GlcNAcbeta1,6[Neu5NAcalpha2,3-Galbeta1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for alpha2,3 linkage. In contrast, the linear alpha2,3-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for alpha2,3 linkage. The branched alpha2,3- and alpha2,6-sialylated O-glycoform Neu5NAcalpha2,3-Galbeta1,3[Neu5NAcalpha2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for alpha2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.