Substituted aminopyrimidine inhibitors have recently been introduced as anti-tuberculosis agents. These inhibitors show impressive activity against PknB, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the PknB enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent PknB inhibitors in the future. Here, molecular dynamics (MD) simulations, in conjunction with MM-PBSA binding free energy analysis, were employed to gain insight into the complex structures of the PknB inhibitors and their binding energetics. The complex structures obtained by the MD simulations show binding free energies in good agreement with experiment. The detailed analysis of MD results shows that Glu93, Val95 and Leu17 are key residues responsible to the binding of the PknB inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of PknB inhibitors with highly increased antagonistic activity.This article is protected by copyright. All rights reserved.