West Nile virus (WNV) requires programmed -1 ribosomal frameshifting for translation of the viral genome. The efficiency of WNV frameshifting is among the highest known. However, it remains unclear why WNV exhibits such a high frameshifting efficiency. Here, we employed dual-luciferase reporter assays in multiple human cell lines to probe the RNA requirements for highly efficient frameshifting by the WNV genome. We find that both the sequence and structure of a predicted RNA pseudoknot downstream of the slippery sequence-the codons in the genome on which frameshifting occurs-are required for efficient frameshifting. We also show that multiple proposed RNA secondary structures downstream of the slippery sequence are inconsistent with efficient frameshifting. We also find that the base of the pseudoknot structure likely is unfolded prior to frameshifting. Finally, we show that many mutations in the WNV slippery sequence allow efficient frameshifting, but often result in aberrant shifting into other reading frames. Mutations in the slippery sequence also support a model in which frameshifting occurs concurrent with or after ribosome translocation. These results provide a comprehensive analysis of the molecular determinants of WNV-programmed ribosomal frameshifting and provide a foundation for the development of new antiviral strategies targeting viral gene expression.