Lipid droplets (LDs) are multi-functional organelles that are found in some prokaryotic cells and all eukaryotic cells. LDs are mainly involved in lipid storage and metabolism with their surface proteins as well as size change. Although several proteins have been identified to mediate LD fusion and fission, the molecular mechanism how cells control LD size still remains to be elucidated. Without other membranous organelles, prokaryotic cells are more suitable for studying LD size regulation than eukaryotic cells. Therefore, LD dynamics are investigated using bacteria Rhodococcus sp. RHA1 as a model system. Our previous work shows that a major LD-associated protein, microorganism lipid droplet small (MLDS) affects LD size in RHA1. Knockout of MLDS results in the formation of supersized LDs without changing the amount of triglyceride. However, how the bacteria regulate MLDS expression remains elusive. Here, we report a novel gene is localized in the same operon with MLDS. Based on bioinformatics analysis, the protein was predicted to be a transcriptional regulator. Using RT-qPCR analysis and confocal microscopy observation, we found that overexpression of the protein reduced MLDS expression level and induced large LD formation. Therefore, we named the protein regulator of MLDS (RMLDS). Our data suggest that RHA1 regulate MLDS expression and LD size by RMLDS.