High pressure homogenization (HPH) is a process that can alter the conformation and functionality of enzymes. The objectives of this study were: (i) evaluate the influence of HPH up to 190 MPa on the proteolytic and milk-clotting activities and stability of four milk-clotting enzymes, (ii) monitor the coagulation process by rheological assays and (iii) evaluate the gel development for 24 hours analyzing proteolysis, syneresis, rheological and microstructural behavior. The evaluations were performed by comparing the results between the processed and non-processed enzymes. The calf rennet processed at 190 MPa decreased 52 % its proteolytic activity, increased the rate of milk-clotting and a more consistent gel was formed. The evaluation of the gel for 24 hours indicated the formation of a protein network with lower proteolysis, higher syneresis, higher consistency and lower porosity. After processing at 150 MPa adult bovine rennet showed a reduction proteolytic activity, increase activity and stability of milk-clotting, higher milk-clotting rate and formed more consistent gels. This gel was more compact, firm and higher whey separation of protein matrix during the 24 hours of evaluation.The fungal protease from Rhizomucor miehei was the most resistant enzyme to the HPH process, showing minimal or no change in proteolytic activity and milk coagulation when processed up to 190 MPa at different concentrations and multiple consecutive processes. However, in the rheological evaluation of milk coagulation using fungal protease homogenized to 190 MPa for up 3 cycles or when homogenized in a solution with a concentration of 20% observed increase in the consistency of the gel. For porcine pepsin, changes on proteolytic activity and milk coagulation were only observed during storage, with reduction of proteolytic activity and an increase on the milk-clotting activity for the enzyme processed at 150 MPa. However, this enzyme promoted a faster coagulation of milk forming more consistent gel immediately after the processing by HPH. During the observation of this gel for 24 hours, this was more compact, firm, less porous and more release of whey of the protein matrix. Overall it was concluded that the viii highest applied pressures (150 MPa and 190 MPa) positively affected the enzymes with reduced nonspecific proteolytic activity and increased milk-clotting activity, with consequent formation of gels with lower levels of proteolysis, which favors the maintenance of a network of protein rigid, firm and cohesive. Thus, it is concluded that HPH is a promising process that can be applied as a technology to improve the hydrolytic characteristics of milk coagulating enzymes, especially to reduce proteolytic activity and increase the milk-clotting activity. Furthermore, the lower proteolysis in the gel may result in an extension of the shelf life of fresh cheese, by possibly reducing the formation of bitterness flavor.