Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol.