Abstract: The gene, ms6174, encoding a transcription factor which affected the formation of mycobacterial biofilm was identified in Mycobacterium smegmatis using transposition insertion mutant library screening as described previously. Subsequently, electrophoretic mobility shift assay（EMSA） and β-galactosidase activity assays confirmed that Ms6147 negatively regulated the ms6147 operon. We found that Ms6174 has characteristics of a nucleoid-associated protein by EMSA, DNase I digestion, and fluorescence co-localization assays. Namely, Ms6174 is a novel nucleoid-associated protein in M. smegmatis. RNA-Seq data analysis showed that 682 genes expression were significantly changed in the ms6174 knockout strain compared with that of the wild-type strain, of which 387 genes were up-regulated and 295 genes were down-regulated. Among them, 142 lipid metabolism related genes were changed. By analysis of the colony morphology and the formation of air-liquid interface pellicles, we found that wrinkles and air-liquid interface pellicles of the ms6174 knockout strain significantly increased compared to that of the wild-type strain. Therefore, it is found that Ms6174 negatively regulate mycobacterial biofilm formation, and it is inferred that the formation of mycobacterial biofilm is regulated by the expression of genes involved in lipid metabolism. The findings identified a new nucleoid-associated protein in mycobacteria and deepened our understanding of the regulatory mechanisms of mycobacterial biofilm formation.