Some legume species of the Crotalaria genus are specifically nodulated by methylotrophic bacteria belonging to the Methylobacterium nodulans species. The feature of this symbiotic bacterium is its ability to oxidize methanol, a property based on the presence of a methanol dehydrogenase enzyme. Despite a good knowledge of this property and its implication in symbiosis, the molecular dialogue between M. nodulans and crotalaria podocarpa leading to symbiosis is largely unknown, except the presence of a nodA nodulation gene in the genome of M. nodulans ORS 2060. To investigate if M. nodulans ORS 2060 produces Nod factors, molecules considered as the major bacteria-to-plant signals essential for the establishment of rhizobia–legume symbiosis, we identified and sequenced a nodDABCUIJHQ cluster from a genomic library of ORS 2060. Phylogenetic analyses of nod genes revealed that M. nodulans ORS 2060 form a branch together with Burkholderia tuberum STM678 and a strain of Methylobacterium sp. (4-46) isolated from Lotononis, and distinct from all the other rhizobia. To analyse the regulation of ORS 2060 nod genes, we constructed a nodA–LacZ promoter fusion to monitor the nod gene expression with various flavonoids. The flavone apigenin was found to be the strongest inducer of nod gene expression in M. nodulans ORS 2060. This latter flavonoid was used to induce ORS 2060, and Nod factors were purified by high-performance liquid chromatography (HPLC) and further characterized by mass spectrometry. One major Nod factor structure was identified as a pentamer of chitin substituted by C18:1 or C16:0 acyl chains on the non-reducing end and 6-O-sulphated on the other end, suggesting a classic symbiotic dialogue between M. nodulans and C. podocarpa.