Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.