The rapid emergence of microbial drug resistance indicates the urgent need for development of new antimicrobial agents. Bacterial cell division machinery is considered as a promising antimicrobial target. Totarol is a naturally existing diterpenoid, which has the ability to restrain bacterial growth by perturbing the cell division. The present study was conducted to investigate the proteomic alterations in Bacillus subtilis as a consequence of totarol treatment to decipher its mechanism of action and possible molecular targets. Cellular proteome of the totarol treated B. subtilis AH75 strain was analyzed by using multiple complementary proteomic approaches. After the drug treatment, 12, 38 and 139 differentially expressed (1.5 fold change) proteins were identified using 2-DE, DIGE and iTRAQ analyses, respectively. In silico functional analysis of the identified differentially expressed proteins indicated a possible effect of totarol on the central metabolism for energy production, heme biosynthesis and chemotaxis. Interestingly, the primary dehydrogenases, which play a vital role in generating the reducing equivalent, were found to be repressed after totarol treatment indicating an apparent metabolic shutdown. Consequently, multiple cellular assays including resazurin assay and FACS analysis of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining confirmed the effect of totarol on respiratory activity and cellular metabolism. Biological significance: The exact mechanism of action of totarol is still unclear and further investigations are essential to identify the molecular/cellular targets of this potential antimicrobial agent. The present study demonstrates the application of differential proteome to decipher the mechanism of action and molecular targets of totarol in B. subtilis. Our quantitative proteome analysis revealed that totarol induced alterations in the expression levels of 139 proteins (1.5 fold change and ≥. 2 peptides) in B. subtilis. Findings obtained from this study indicate that totarol treatment leads to metabolic shutdown by repressing the major central metabolic dehydrogenases in B. subtilis. In addition, expression levels of universal chaperone proteins, heme biosynthesis, and ribosomal proteins were found to be altered, which caused the filamentation of the bacteria. To the best of our knowledge, this is the foremost inclusive investigation describing totarol induced alterations in B. subtilis proteome and diverse physiological processes. We anticipate that this in depth proteomic study may contribute to a better understanding of the mode of action of totarol and its primary molecular and cellular targets. © 2014 .