The long noncoding RNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether ENOD40 was required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 express from chromosome 5 (chr5) (AhENOD40-1) and chr15 (AhENOD40-2) during symbiosis and RNA interference of these transcripts drastically affected nodulation, indicating the importance of ENOD40 in A. hypogaea. Furthermore, we demonstrated several distinct characteristics of ENOD40. (i) Natural antisense transcript (NAT) of ENOD40 was detected from the AhENOD40-1 locus (designated as NAT-AhDONE40). (ii) Both AhENOD40-1 and AhENOD40-2 had two exons, whereas NAT-AhDONE40 was monoexonic. Reverse-transcription quantitative PCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus and their expression increased with the progress of symbiosis. (iii) RNA pull-down fromwhole cell extracts of infected roots at 4 days postinfection indicated NAT-AhDONE40 to interact with the SET (Su(var)3-9, enhancer of Zeste and Trithorax) domain containing absent small homeotic disc (ASH) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and electrophoretic mobility shift assay. (iv) Chromatin immunoprecipitation assays indicate deposition of ASHR3-specific histone marks H3K36me3 and H3K4me3 in both of the ENOD40 loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Because both ASHR3 and ENOD40 from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of DONE40 with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.