Integrative and conjugative elements (ICE) are mobile genetic elements that can excise from bacterial chromosomes and horizontally transfer via conjugation. The ICE of Mesorhizobium loti R7A, ICEMlSymR7A, is an element that transfer to other mesorhizobia in soils, converting recipients into nitrogen-fixing symbionts of legumes. ICE excision is catalysed by a site-specific recombinase IntS, which recombines direct-repeat attachment sites attL and attR that flank the chromosomally-integrated ICE. Recombination produces a circular excised ICE molecule containing an attP site and attB in the mesorhizobial chromosome. The excision reaction in ICEMlSymR7A requires a predicted DNA-binding protein RdfS. We used "mini-ICE" plasmids containing various attP regions to define the minimal region able to facilitate IntS-mediated recombination in ICEMlSymR7A. Purified RdfS was then used to define the RdfS binding sites within attP using surface-plasmon-resonance (SPR)-based DNA footprinting. A single 40-bp region exhibited strong binding to RdfS. This DNA region was used in Small-Angle X-ray Scattering (SAXS) experiments to determine the size and shape of the RdfS-attP molecule. RdfS appears to form a tetramer across the entire 40-bp DNA region in a two-fold symmetry. Understanding RdfS DNA-binding activity unravels its multifaceted role in ICE recombination, transcriptional regulation and conjugative activity.