A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca2+-binding sites (Ca1–Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca2+-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5°C in T1/2. It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5°C in T1/2 but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.