Endo-1,4-beta-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R. albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals. The crystallographic structure of Xyn10A is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the best similarity was found with the Clostridium thermocellum (C. thermocellum) N-terminal endo-1,4-beta-D-xylanase 10 b. Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the -1 subsite of Xyn10A in the C-4(1) (chair) and (2)So (skew boat) ground state conformations. According to the simulations and free energy analysis, Xyn10A binds the substrate with the -1 sugar in the (2)So conformation 39.27 kcal.mol(-1) tighter than the substrate with the sugar in the C-4(1) conformation. According to the Xyn10A-(2)So Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite -1. The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the -1 sugar subsite proceeds from the C-4(1) conformation through (2)So to the transition state. MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite -1 may an important residue for substrate binding. Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.