Pyrazinamide is an essential first‐line antitubercular drug which plays pivotal role in tuberculosis treatment. It is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme for conversion into pyrazinoic acid (POA). POA is known to target ribosomal protein S1 (RpsA), aspartate decarboxylase (PanD), and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against pyrazinamide. We have constructed and validated 3D models of the native and Δ438A mutant form of RpsA protein. RpsA protein variants were then docked to POA and long range molecular dynamics simulations were carried out. Per residue binding free‐energy calculations, free‐energy landscape analysis, and essential dynamics analysis were performed to outline the mechanism underlying the high‐level PZA resistance conferred by the most frequently occurring deletion mutant of RpsA. Our study revealed the conformational modulation of POA binding site due to the disruptive collective modes of motions and increased conformational flexibility in the mutant than the native form. Residue wise MMPBSA decomposition and protein‐drug interaction pattern revealed the difference of energetically favorable binding site in the wild‐type (WT) protein in comparison with the mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the WT complex than the mutant form. Our study provides mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis. Mutations in mycobacterial ribosomal protein S1 (RpsA) have been reported to cause resistance against pyrazinamide. Our study revealed the conformational modulation of drug‐binding site due to the disruptive collective modes of motions and increased conformational flexibility in the RpsA mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the wild‐type complex than the mutant form. We have provided the mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.