RNA interference (RNAi) is the biological mechanism that allows targeted gene knockdown through the addition of exogenous short-interfering RNAs (siRNAs) to cells and organisms. RNAi has revolutionized cell biology and holds enormous potential for human therapy. One of the major challenges facing RNAi as a therapy is achieving efficient and nontoxic delivery of siRNAs into the cell cytoplasm, since their highly anionic character precludes their passage across the cell membrane unaided. Herein, we report a novel fusion protein between the tombusviral p19 protein, which binds siRNAs with picomolar affinity, and the “TAT” peptide (RKKRRQRRRR), which is derived from the transactivator of transcription (TAT) protein of the human immunodeficiency virus and acts as a cell-penetrating peptide. We demonstrate that this fusion protein, 2x-p19-TAT, delivers siRNAs into the cytoplasm of human hepatoma cells where they elicit potent and sustained gene knockdown activity without toxic effects.