KNOTTED1‐like homeobox (KNOX) proteins are homeodomain transcription factors that play an important role in plant development. In the shoot apical meristem KNOX genes are responsible for the activation of isopentenyl transferase genes (IPTs) involved in cytokinin biosynthesis. Plant hormones play a key role in shoot apical meristem maintenance. Cytokinin is a well‐known regulator of nodule development playing a key role in the regulation of cell division during nodule primordium formation. Moreover, the activation of IPTs genes has been shown to take place during nodulation. We hypothesize that KNOX TFs may regulate nodule development and activate cytokinin biosynthesis upon nodulation. Therefore, we analyzed the expression of different MtKNOX genes in Medicago truncatula upon nodule development. Activation of MtKNOX3 expression in response to inoculation with Sinorhizobium meliloti was found. MtKNOX3 promoter activity as revealed with a pMtKNOX3:GUS reporter fusion was observed in developing nodule primordia. MtKNOX3 ectopic expression caused the formation of nodule‐like structures on transgenic roots without bacterial inoculation, a phenotype similar to the one previously described for legumes with constitutive activation of the cytokinin receptor. MtKNOX3 RNAi resulted in the downregulation of the type‐A cytokinin response gene MtRR4, as well as the MtIPT3 gene and the LONELYGUY2 (MtLOG2) gene involved in cytokinin activation. To summarize, our data suggest that MtKNOX3 is involved in symbiotic nodule development via the regulation of cytokinin production in M. truncatula.