Histo-blood group antigens (HBGAs) have been recognized as receptors/co-receptors for human norovirus (HuNoV) and Tulane virus (TV). It has been reported that exposure of TV to free HBGAs in human saliva (HS) completely abolished viral binding and infectivity in LLC-MK2 cells as measured by ELISA. This is contrary to the clinical observations that HuNoV exposure to free HBGAs in HS in vivo does not diminish the viral infectivity. In this study, we tested whether pre-exposure of TV to free HBGA in HS inhibited viral binding and subsequent infection of cell culture by a more sensitive in vitro ligand based molecular assay and a cell culture-based qRT-PCR assay. In an in vitro HS-based binding assay, pre-incubation HS resulted in an 88% reduction in viral binding. In an in vivo cell culture-based replication assay, pre-exposure of TV to HS resulted in a significantly reduced viral replication only at earlier stage of infection. A 77.8%, 88.3%, 44.5%, and 14.8% inhibition of TV replication at 24, 48, 72, and 96 hpi., respectively. Our results suggest that free HBGAs in HS cannot provide adequate protection to prevent infection of HuNoV. This new hypothesis better explains the differences between clinical and past experimental observations, and why HBGA-secreting hosts are still susceptible to infection by HuNoV.