RNA modifications play important roles in RNA structures and regulation of gene expression and translation. We report the first RNA modification on the phosphate, the RNA phosphorothioate (PS) modification, discovered in both prokaryotes and eukaryotes. The PS modification is also first reported on nucleic acids of eukaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By knocking out the DndA gene in E. coli, we show the Dnd clusters that regulate DNA PS modification may also play roles in RNA PS modification. We also show that the GpsG modification locates on rRNA in E. coli, L. lactis, and HeLa cells, and it is not detected in rRNA-depleted total RNAs from these cells.