Background: Conjugative spread of antibiotic resistance and virulence genes in bacteria constitutes an important threat to public health. Beyond the well-known conjugative plasmids, recent genome analyses have shown that integrative and conjugative elements (ICEs) are the most widespread conjugative elements, even if their transfer mechanism has been little studied until now. The initiator of conjugation is the relaxase, a protein catalyzing a site-specific nick on the origin of transfer (oriT) of the ICE. Besides canonical relaxases, recent studies revealed non-canonical ones, such as relaxases of the MOB T family that are related to rolling-circle replication proteins of the Rep_trans family. MOB T relaxases are encoded by ICEs of the ICESt3/ICEBs1/Tn916 superfamily, a superfamily widespread in Firmicutes, and frequently conferring antibiotic resistance. Results: Here, we present the first biochemical and structural characterization of a MOB T relaxase: the RelSt3 relaxase encoded by ICESt3 from Streptococcus thermophilus. We identified the oriT region of ICESt3 and demonstrated that RelSt3 is required for its conjugative transfer. The purified RelSt3 protein is a stable dimer that provides a Mn 2+-dependent single-stranded endonuclease activity. Sequence comparisons of MOB T relaxases led to the identification of MOB T conserved motifs. These motifs, together with the construction of a 3D model of the relaxase domain of RelSt3, allowed us to determine conserved residues of the RelSt3 active site. The involvement of these residues in DNA nicking activity was demonstrated by targeted mutagenesis. Conclusions: All together, this work argues in favor of MOBT being a full family of non-canonical relaxases. The biochemical and structural characterization of a MOBT member provides new insights on the molecular mechanism of conjugative transfer mediated by ICEs in Gram-positive bacteria. This could be a first step towards conceiving rational strategies to control gene transfer in these bacteria.