Ssu72, an RNA polymerase II C-terminal domain (CTD) phospho-Ser5 (pSer5) phosphatase, was recently reported to have pSer7 phosphatase activity as well. We report here the crystal structure of a ternary complex of the N-terminal domain of human symplekin, human Ssu72, and a 10-mer pSer7 CTD peptide. Surprisingly, the peptide is bound in the Ssu72 active site with its backbone running in the opposite direction compared with a pSer5 peptide. The pSer7 phosphatase activity of Ssu72 is ∼4000-fold lower than its pSer5 phosphatase activity toward a peptide substrate, consistent with the structural observations.