Antimicrobial resistance (AMR) poses a threat to global health and the economy. Rifampicin-resistant Mycobacterium tuberculosis accounts for a third of the global AMR burden. Gaining the upper hand on AMR requires a deeper understanding of the physiology of resistance. AMR often results in a fitness cost in absence of drug. Identifying the molecular mechanisms underpinning this cost could help strengthen future treatment regimens. Here, we used a collection of M. tuberculosis strains providing an evolutionary and phylogenetic snapshot of rifampicin resistance, and subjected them to genome-wide transcriptomic and proteomic profiling to identify key perturbations of normal physiology. We found that the clinically most common rifampicin resistance-conferring mutation RpoB Ser450Leu imparts considerable gene expression changes, many of which are mitigated by the compensatory mutation in RpoC Leu516Pro. However, our data also provide evidence for pervasive epistasis: the same resistance mutation imposed a different fitness cost and functionally distinct changes to gene expression in genetically unrelated clinical strains. Finally, we report a likely post-transcriptional modulation of gene expression that is shared in most of the tested strains carrying RpoB Ser450Leu, resulting in an increased abundance of proteins involved in central carbon metabolism. These changes contribute to a more general trend, in which the disruption of the composition of the proteome correlates with the fitness cost of the RpoB Ser450Leu mutation in different strains.