A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an alpha/beta hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca2+-induced thermostabilization and activation of enzymes have been well explored in alpha-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca2+ enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190(S226P)) in the Ca2+-bound and free states at 1.75 and 1.45 resolution, respectively. Based on the crystallographic data, a Ca2+ ion was coordinated by four residues within loop regions (the Ca2+ site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca2+ to Cut190(S226P) induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca2+ not only stabilized a region that is flexible in the Ca2+-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca2+-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190(S226P) is activated by a conformational change in the active-site sealing residue, F106.