Summary The ta‐siRNA pathway is required for lateral organ development, including leaf patterning, flower differentiation and lateral root growth. Legumes can develop novel lateral root organs – nodules – resulting from symbiotic interactions with rhizobia. However, ta‐siRNA regulation in nodule formation remains unknown. To explore ta‐siRNA regulation in nodule formation, we investigated the roles of REL3, a key component of TAS3 ta‐siRNA biogenesis, during nodulation in Lotus japonicus. We characterized the symbiotic phenotypes of the TAS3 ta‐siRNA defective rel3 mutant, and analyzed the responses of the rel3 mutant to auxin and ethylene in order to gain insight into TAS3 ta‐siRNA regulation of nodulation. The rel3 mutant produced fewer pink nitrogen‐fixing nodules, with substantially decreased infection frequency and nodule initiation. Moreover, the rel3 mutant was more resistant than wild‐type to 1‐naphthaleneacetic acid (NAA) and N‐1‐naphthylphthalamic acid (NPA) in root growth, and exhibited insensitivity to auxins but greater sensitivity to auxin transport inhibitors during nodulation. Furthermore, the rel3 mutant has enhanced root‐specific ethylene sensitivity and altered responses to ethylene during nodulation; the low‐nodulating phenotype of the rel3 mutant can be restored by ethylene synthesis inhibitor L‐α‐(2‐aminoethoxyvinyl)‐glycine (AVG) or action inhibitor Ag+. The REL3‐mediated TAS3 ta‐siRNA pathway regulates nodulation by integrating ethylene and auxin signaling.