Microorganism lipid droplet small regulator (MLDSR) is a transcriptional regulator of the major lipid droplet (LD)‐associated protein MLDS in Rhodococcus jostii RHA1 and Rhodococcus opacus PD630. In this study, we investigated the role of MLDSR on lipid metabolism and triacylglycerol (TAG) accumulation in R. jostii RHA1 at physiological and molecular levels. MLDSR gene deletion promoted a significant decrease of TAG accumulation, whereas inhibition of de novo fatty acid biosynthesis by the addition of cerulenin significantly repressed the expression of the mldsr‐mlds cluster under nitrogen‐limiting conditions. In vitro and in vivo approaches revealed that MLDSR‐DNA binding is inhibited by fatty acids and acyl‐CoA residues through changes in the oligomeric or conformational state of the protein. RNAseq analysis indicated that MLDSR not only controls the expression of its own gene cluster but also of several genes involved in central, lipid, and redox metabolism, among others. We also identified putative MLDSR‐binding sites on the upstream regions of genes coding for lipid catabolic enzymes and validated them by EMSA assays. Overexpression of mldsr gene under nitrogen‐rich conditions promoted an increase of TAG accumulation, and further cell lysis with TAG release to the culture medium. Our results suggested that MLDSR is a fatty acid‐responsive regulator that plays a dual role in cells by repression or activation of several metabolic genes in R. jostii RHA1. MLDSR seems to play an important role in the fine‐tuning regulation of TAG accumulation, LD formation, and cellular lipid homeostasis, contributing to the oleaginous phenotype of R. jostii RHA1 and R. opacus PD630.