Delta(5)-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroids to their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction follows a two-step general acid-base mechanism starting with Asp38-CO2- mediated proton abstraction from a sp(3)-hybridized carbon atom, alpha to carbonyl group, providing a dienolate intermediate. In the second step, Asp38-CO2H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details of the mechanism have been highly controversial despite several experimental and computational studies of this enzyme. The general acid-base catalysis has been proposed to involve either a catalytic diad or a cooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecond molecular dynamics (MD) simulation of enzyme bound natural substrate (E-S) and enzyme bound intermediate ((EIn)-In-.) solvated in a TIP3P water box. The final coordinates from our MD simulation strongly support the cooperative hydrogen bond mechanism. The MD simulation of E-S and E-In shows that both Tyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogen bonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on (ES)-S-. --> (EIn)-In-.. We also observe dynamic motions of water moving in and out of the active site in the (ES)-S-. structures. This free movement of water disappears in the (EIn)-In-. structures. The active site is shielded by hydrophobic residues, which come together and squeeze out the waters from the active site in the (EIn)-In-. complex.