Abstract: To study the effect of ultrahigh pressure treatment on papain activity and the molecular mechanism, papain solutions were treated with 0.1, 200 and 600 MPa to determine the changes in activity. And molecular simulation of papain at these three pressures were performed for 150 ns, the changes of root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), hydrogen bonds, solvent accessible surface area, volume, secondary structure and molecular surface structure was evaluated. The results showed that 200 MPa had an activating effect on papain, while the enzyme activity was reduced by 26.2% at 600 MPa. Molecular simulation showed that high pressure treatment could reduce the fluctuation of protein structure, protein volume, and protein solvent accessible surface area, especially hydrophobic surface area, and make the protein structure more compact. High pressure could destroy the hydrogen bond between enzyme proteins and increase the content of β-sheet, while decrease the α-helix obviously at 600 MPa. The activation of the enzyme at 200 MPa may be related to the larger binding pocket in the active center of the enzyme at this pressure, which made it easier to bind to the target protein. The partial inhibition of enzyme activity under 600 MPa was related to the destruction of enzyme structure under this pressure, for example, the destruction of α-helix and hydrogen, the reduction of hydrophobic surface area and protein volume. The molecular mechanism of papain activity change under high pressure was clarified.