Many species of Grain-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a "lid", a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the "lid" in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.