We demonstrate that a virally encoded yeast ‘killer’ toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted α/β heterodimer that kills sensitive yeasts in a receptor‐mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose β‐C‐terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Δerd2) or mutants defective in Golgi‐to‐ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site‐directed mutagenesis further indicated that the toxin's β‐HDEL motif ensures retrograde transport, although in a toxin‐secreting yeast the β‐C‐terminus is initially masked by an R residue (β‐HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high‐level secretion of a biologically inactive protein incapable of re‐entering the secretory pathway. Finally, we present evidence that ER‐to‐cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.