Translational readthrough (TR), the elongation of the polypeptide chain by the ribosome beyond the stop codon, was initially observed for viral RNA and more recently reported for yeast and animal transcripts. TR modulates the protein output and diversifies the proteome 1–5. Here, we report that the expression of a TR isoform of Argonaute 1 (AGO1x) is strongly correlated with the proliferative potential of human breast tumors. In contrast to the canonical AGO1 isoform, AGO1x localizes to the nucleus of human cells. Loss of AGO1x impairs the growth of rapidly dividing cells and leads to accumulation of double stranded RNAs with consequent induction of the interferon response and apoptosis. Our data thus uncover a novel function for a mammalian member of the Argonaute protein family, beyond the miRNA effector pathway. As the specific targeting of the AGO1x strongly impacts the proliferation of cancer cells, our study provides a new approach to interfering with tumor growth.