Triacylglycerol (TAG) is the major storage lipid of mycobacteria. Mycobacterium tuberculosis (Mtb) genome encodes 15 triacylglycerol synthases (Tgs), which are speculated to differ in substrate preference, suggesting specific physiological roles. In this study, we investigated the role of a Tgs, Rv3371, in the context of infection. Rv3371 knock-out (KO) Mtb was attenuated in mice, with corresponding poor fitness inside macrophages. The KO was more sensitive to free long-chain fatty acids, but was more tolerant to oxidative and nitrosative stresses. Enzyme kinetics of Rv3371 showed its preference for propionyl-CoA. Excess propionate in growth medium retarded the growth of the KO more significantly than the wild type and complemented mutant. This suggests an additional role of Rv3371 in reducing toxic levels of propionate in Mtb by synthesising propionyl TAG. Moreover, chemical inhibition of methylcitrate cycle caused a decrease in methyl-branched lipids in the KO. Overall, the results suggest a role of Rv3371 in Mtb survival in the host through its roles beyond TAG storage.