Enzymatic reactions with supercritical carbon dioxide (SC-CO2) have received increased attention during the last decade. The objective of this study was to alter the activity and stability of Bacillus sp. protease under SC-CO2 conditions. The activity and stability of protease were evaluated according to the effects of operational variables, temperature (28–80°C), pressure (60–300bar), CO2 flow (2–10g/min) and process duration (60–180min). Best conditions were identified as 300bar, 54°C, 6g/min CO2 flow and 120min of process time efficient 54.4% (417.50μmol/ml/min) as against to the untreated enzyme. Activity enhancement was observed with the raise of pressure while low flow rates decreased the activity by 5.1% with a flow rate of 2g/min under supercritical conditions. Potential mechanisms for pressure stimulated activation and stabilization were investigated by NMR, SEM, FTIR, SDS-PAGE and XPS analyses. As a result, applications of SC-CO2 medium for enzymatic processes are expected to become sustainable and important with economical synthetic and environmentally friendly protocols.