The synthesis of an RNA dinucleotide (IspU) containing a 3’-S-phosphorothiolate linkage is described. The compound is prepared from 9-(3-deoxy-3-iodo-beta-D-xylofuranosyl)hyperanthine with installation of the phosphorothiolate group via an Arbusov reaction and protection of the ribose 2’-hydroxyl as a silyl ether. IspU is found to be a substrate for several enzymes including T4 polynucleotide kinase, snake venom phosphodiesterase, and ribonuclease T-2. Base-catalyzed cleavage of the dinucleotide is accelerated (similar to 2000-fold) relative to that of the phosphate-linked compound IpU. Product characterization and kinetic analysis show that IspU is cleaved through the same mechanism as IpU. The observed rate acceleration is argued to reflect stabilization of the anionic transition state by the polarizable sulfur atom.