The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation， phosphoryl-Ser (pSer) is gradually dephosphorylated by CTD phosphatases， whereas Ser phosphorylation accumulates. Using MS， X-ray crystallography， protein engineering， and immunoblotting analyses， here we investigated the structure and function of SSU72 homolog， RNA polymerase II CTD phosphatase (Ssu72， from )， an essential CTD phosphatase that dephosphorylates pSer at the transition from elongation to termination， to determine the mechanism by which Ssu72 distinguishes the highly similar pSer and pSer CTDs. We found that Ssu72 dephosphorylates pSer effectively but only has low activities toward pSer and pSer The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the configuration， forming a tight β-turn for recognition by Ssu72. We also noted that residues flanking the SP motif， such as the bulky Tyr next to Ser， prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr further prohibited Ssu72 binding to pSer and thereby prevented untimely Ser dephosphorylation. Our results reveal critical roles for Tyr in differentiating the phosphorylation states of Ser/Ser of CTD in RNA polymerase II that occur at different stages of transcription.