The objectives of this research were to extract, partially purify, and characterize proteases from hepatopancreas of Cardisoma guanhumi. Enzymes from the hepatopancreas of this semi-terrestrial crab were purified in three steps: heat treatment (45^oC during 30 min), ethanol fractionation (10% to 90% of saturation) and ultrafiltration (50kDa membrane). Protein content and proteolytic activity were determined for each fraction. The fraction presenting the highest specific activity was submitted to physico-chemical characterization (thermal stability, optima pH and temperatures, and metalic ions effects). SDS-PAGE was also carried out to evaluate the purification steps. One major band was observed by SDS-PAGE (70.7 kDa) on 10% fraction. This preparation presented an optimum temperature of 60^oC (using BApNA as a substrate); it was stable for 30 min at 60^oC and showed high proteolytic activity in the pH range from 7.0 to 10.0, with a slightly higher activity at 8.5. The protease presented moderate inhibition by Cd²⁺, Cu²⁺, Hg²⁺, Zn²⁺ and Mn²⁺. On the other hand, Ca²⁺ and Al³⁺ were capable to increase the proteolytic activity of that fraction. Inhibition by PMSF and TLCK provided additional evidences that this activity can be attributed to a trypsin-like enzyme. The apparent Michaelis-Menten constant (K_m) for BApNA was 0.30 mMol/L. The purification described herein rendered a preparation containing an alkaline thermostable trypsin-like enzyme. These results can also contribute to the understanding of digestive physiology and optimisation of diet formulation in C. guanhumi cultures.