Mycobacterium tuberculosis is the pathogen of tuberculosis which causes about millions people death annually. M. smegmatis, as a type of non-pathogenic strain of Mycobacterium closely related to M. tuberculosis, is the most studied model strain in laboratory. Mycobacterium encodes three types of chromatin proteins, i.e. histone-like protein HU, Lsr2 and integration host factor (IHF). To investigate the functional roles of IHF in chromosomal DNA organization, we expressed and purified IHF protein from M. smegmatis (MsIHF) in Escherichia coli, and analyzed the effects of MsIHF on DNA topology in vitro. MsIHF exists as a stable homodimer in solution. MsIHF exhibits preferred binding to negatively supercoiled DNA rather than linear or relaxed DNA. This protein is also capable of constraining negative DNA supercoils. Further analyses showed modulations of topoisomerase activities by MsIHF in vitro. MsIHF obviously inhibits the relaxation of supercoiled DNA by topoisomeraseⅠ from E. coli. By contrast, this protein slightly stimulates E. coli gyrase to introduce negative supercoils into relaxed DNA. These data suggests that MsIHF may alter the topological structure of chromosomal DNA through modulation of the activities of topoisomerases, and therefore regulates the organization of chromosome.