Abstract:DNA phosphorothioate modification was the first reported physiological modification in the DNA backbone. Five putative proteins encoded by the five-member dndABCDE gene cluster replaced the non-bridging oxygen in the sugar-phosphate backbone with a sulfur. Phosphorothioate modification occurrs in sequence-selective and Rp stereo-specific manner in diverse bacterial stains. In recent years, researchers have made systemic achievements in this area. To have a comprehensive understanding of this unusual modification, we reviewed the discovery and research progress in DNA phosphorothioate modification and also discussed opportunities and challenges in the future.