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–O5′′ 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 N 1‑methylpseudouridine (m1Ψ) 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–O5′′ bond on the 3′ side of Ψ and m1Ψ. We find that these enzymes catalyze the cleavage of UpA up to 10‑fold more efficiently than the cleavage of ΨpA or m1ΨpA. 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 m1Ψ 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 pK a of the 2′‑hydroxy group of U and enhances its nucleophilicity toward the P–O5′′ bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does m1ΨpA. Our findings inform the continuing development of RNA-based vaccines and therapeutic agents.