Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.