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In clinical uses, RNA must maintain its integrity in serum that contains ribonucleases (RNases), especially RNase 1, which is a human homolog of RNase A. These omnipresent enzymes catalyze the cleavage of the P-O bond on the 3' side of pyrimidine residues. Pseudouridine (Ψ) is the most abundant modified nucleoside in natural RNA. The substitution of uridine (U) with Ψ or -methylpseudouridine (mΨ) reduces the immunogenicity of mRNA and increases ribosomal translation, and these modified nucleosides are key components of RNA-based vaccines. Here, we assessed the ability of RNase A and RNase 1 to catalyze the cleavage of the P-O bond on the 3' side of Ψ and mΨ. We find that these enzymes catalyze the cleavage of UpA up to 10-fold more efficiently than the cleavage of ΨpA or mΨpA. X-ray crystallography of enzyme-bound nucleoside 2',3'-cyclic vanadate complexes and molecular dynamics simulations of enzyme·dinucleotide complexes show that U, Ψ, and mΨ bind to RNase A and RNase 1 in a similar manner. Quantum chemistry calculations suggested that the higher reactivity of UpA is intrinsic, arising from an inductive effect that decreases the p of the 2'-hydroxy group of U and enhances its nucleophilicity toward the P-O bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does mΨpA. Our findings reveal a new role for natural pseudouridine residues and inform the continuing development of RNA-based vaccines and therapeutic agents.