Over the past 5 years, there has been a renewed interest in finding new compounds with anti-TB action. Colistin methanesulfonate or polymyxin E, is a possible anti-TB drug candidate, which may in future be used either alone or in combination to the current 6 month “directly observed treatment short-course” (DOTS) regimen. However its mechanism of action has to date not yet been fully explored, and only described from a histological and genomics perspective. Considering this, we used a GCxGC-TOFMS metabolomics approach and identified those metabolite markers characterising Mycobacterium tuberculosis (Mtb) cultured in the presence of colistin methanesulfonate, in order to better understand or confirm its mechanism of action. The metabolite markers identified indicated a flux in the metabolism of the colistin methanesulfonate treated Mtb towards fatty acid synthesis and cell wall repair, confirming previous reports that colistin acts by disrupting the cell wall of mycobacteria. Accompanying this, is a subsequently elevated glucose uptake, since the latter now serves as the primary energy substrate for the upregulated glyoxylate cycle, and additionally as a precursor for further fatty acid synthesis via the glycerolipid metabolic pathway. Furthermore, the elevated concentrations of those metabolites associated with pentose phosphate, valine, threonine, and pentanediol metabolism, also confirms a shift towards glucose utilization for energy production, in the colistin methanesulfonate treated Mtb.