RicinA‐chain (RTA) depurinates the sarcin–ricin loop of 28S ribosomalRNAand inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction ofRTAwith the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers ofP1 andP2 bound toP0, whereas the yeast stalk consists of two different heterodimers,P1α–P2β andP2α–P1β, bound toP0.RTAexhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction ofRTAwith human ribosomes and subsequent depurination of the sarcin–ricin loop. Using small interferingRNA‐mediated knockdown ofP1/P2 expression in human cells, we demonstrated that the depurination activity ofRTAis lower whenP1 andP2 levels are reduced. Biacore analysis showed that ribosomes fromP1/P2‐depleted cells have a reduced ability to bindRTA, which correlates with reduced depurination activity bothin vitroand inside cells.RTAinteracts directly with recombinant humanP1–P2 dimer, further demonstrating the importance of humanP1 andP2 in enablingRTAto bind and depurinate human ribosomes.Structured digital abstractP2, P1 and RTA physically interact by surface plasmon resonance (View interaction)P2, P1 and RTA physically interact by anti bait coimmunoprecipitation (View interaction)