Legume-rhizobial symbiotic nitrogen fixation is the most efficient nitrogen assimilation system in the ecosystem. In the special interaction between organ–root nodules, legumes supply rhizobial carbohydrates for their proliferation, while rhizobials provide host plants with absorbable nitrogen. Nodule initiation and formation require a complex molecular dialogue between legumes and rhizobia, which involves the accurate regulation of a series of legume genes. The CCR4–NOT complex is a conserved multi-subunit complex with functions regulating gene expression in many cellular processes. However, the functions of the CCR4–NOT complex in rhizobia–host interactions remain unclear. In this study, we identified seven members of theNOT4family in soybean and further classified them into three subgroups. Bioinformatic analysis showed thatNOT4sshared relatively conserved motifs and gene structures in each subgroup, while there were significant differences betweenNOT4sin the different subgroups. Expression profile analysis indicated thatNOT4smay be involved in nodulation in soybean, as most of them were induced by Rhizobium infection and highly expressed in nodules. We further selectedGmNOT4-1to clarify the biological function of these genes in soybean nodulation. Interestingly, we found that eitherGmNOT4-1overexpression or down-regulation ofGmNOT4-1by RNAi or CRISPR/Cas9 gene editing would suppress the number of nodules in soybean. Intriguingly, alterations in the expression ofGmNOT4-1repressed the expression of genes in the Nod factor signaling pathway. This research provides new insight into the function of theCCR4–NOTfamily in legumes and revealsGmNOT4-1to be a potent gene for regulating symbiotic nodulation.