Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events including abiotic/biotic stress responses, innate immunity, hormone signaling and cell specificity in plants. The MAPK-mediated stress and ethylene signaling are recently known to be involved in nitogen-fixing symbiotic interactions; however, the biological role of MAPK for nodule development in legume plants is largely unknown. We here elucidated that MtMKK5-MtMPK3/6 cascade negatively regulate the nitrogen fixing nodule formation in Medicago truncatula. MtMKK5, an ortholog of SIMKK, overexpression significantly reduces the nodule formation in M. truncatula roots. MtMKK5 directly activates MtMPK3/6 by phosphorylation on the TEY motif within the activation loop in the cytoplasm, which might link to EFD as a negative regulator for nodule formation. EFD has a putative MAPK phosphorylation Thr residue and could be a target of the activated MtMPK3/6 in the nucleus. Consistently, a MAPK specific inhibitor U0126 enhances nodule formation and confers similar nodule phenotypes to the efd-1 mutant such as lower proliferation and differentiation to symbiotic tissues. Our works thus reveal a key negative signaling module mediated by MtMKK5-MtMPK3/6-EFD for symbiotic nitrogen fixing nodule organogenesis.