Molecular docking, molecular dynamics (MD) simulation and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA)/molecular mechanics Generalized Born surface area (MM-GBSA) analysis are applied to predict the binding mode of two N-substituted pyrrole derivate inhibitors to the hydrophobic pocket in HIV-1 envelope protein gp41. Taking into account the flexibility of the receptor, multiple receptor conformations are used in docking with the ligands, which results in several possible binding modes. MD simulations and MM-PBSA binding energy calculations are performed on all the binding modes to identify the most favorable binding estimate. The MM-PBSA results indicate that the binding is mainly driven by non-polar interactions, while polar interactions determine the orientation of the ligands binding into the target site. Further analysis reveals the key residues and ligand-receptor interactions which contribute significantly to the binding affinity. This study provides useful information for rational design and optimization of N-substituted pyrrole derivatives as HIV-1 fusion inhibitors.