The influenza virus PB1 protein is the core subunit of the heterotrimeric polymerase complex (PA, PB1 and PB2) in which PB1 is responsible for catalyzing RNA polymerization and binding to the viral RNA promoter. Among the three subunits, PB1 is the least known subunit so far in terms of its structural information. In this work, by integrating template-based structural modeling approach with all known sequence and functional information about the PB1 protein, we constructed a modeled structure of PB1. Based on this model, we performed mutagenesis analysis for the key residues that constitute the RNA template binding and catalytic (TBC) channel in an RNP reconstitution system. The results correlated well with the model and further identified new residues of PB1 that are critical for RNA synthesis. Moreover, we derived 5 peptides from the sequence of PB1 that form the TBC channel and 4 of them can inhibit the viral RNA polymerase activity. Interestingly, we found that one of them named PB1(491-515) can inhibit influenza virus replication by disrupting viral RNA promoter binding activity of polymerase. Therefore, this study has not only deepened our understanding of structure-function relationship of PB1, but also promoted the development of novel therapeutics against influenza virus.