The emergence of highly pathogenic avian influenza viruses, such as the H5 subtype, is driven by the insertion of basic amino acids at the hemagglutinin cleavage site. A leading hypothesis suggests these nucleotide insertions result from «polymerase trapping» by a transient RNA pseudoknot (t-loop) formed between the ingoing and outgoing template during viral replication. To demonstrate this mechanism, we determined the cryo-EM structure of a pseudoknot-stalled influenza A polymerase. Because the wild-type t-loop is highly transient, we engineered a functionally optimized 70 nucleotide mini-vRNA genome template designed to precisely stop ongoing RNA synthesis upon formation of the stabilised pseudoknot. In vitro RNA synthesis assays confirmed that the polymerase specifically stalls as intended. Cryo-EM analysis of the stalled state revealed the formation of the predicted double-stranded RNA t-loop distally formed outside the polymerase core, accommodated by the outward swinging of the PB1 beta-ribbon. Furthermore, the structural data captured a two-nucleotide backtracking of the product-template duplex. This backtracking provides direct insight into the insertion mechanism: in a wild-type A:U-rich context, strain likely induces inter-strand slippage, analogous to viral polyadenylation, resulting in the multi-nucleotide insertions as observed in virological studies. These findings provide the first direct visual proof of the t-loop concept, providing mechanistic insights into H5 highly pathogenic avian influenza virus nucleotide insertion. Publication: https://doi.org/10.1126/science.adr6632