AbstractCondensin is the major driving force in the segregation of daughter chromosomes in prokaryotes. Core subunits of condensin belong to the SMC protein family, whose members are characterized by a unique ATPase activity and dimers with a V-shaped structure. The V-shaped dimers might close between head domains, forming a ring structure that can encircle DNA. Indeed, cohesin, which is a subfamily of SMC proteins, encircles double-stranded DNA to hold sister chromatids in eukaryotes. However, the question of whether or not condensin encircles the chromosomal DNA remains highly controversial. Here we report that MukB binds topologically to DNA in vitro and this binding is preferentially single-stranded DNA (ssDNA) rather than double-stranded DNA. The binding of MukB to ssDNA does not require ATP. In fact, thermal energy enhances the binding. The non-SMC subunits MukF and MukE did stimulate the topological binding of MukB, although they hindered DNA-binding of MukB. Recent reports on the distribution of condensin in genomes reveal that actively transcribed genes in yeast and humans are enriched in condensin. In consideration of all these results, we propose that the binding specificity of condensin to chromosome is provided not by the DNA sequence but by the DNA structure, which is ssDNA.