Nodulation inLeguminousspp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specificRhizobiumspp. in the rhizosphere.Medicago sativa(alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa’s relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants withSPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing ofMsSPL9in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed thatMsSPL9regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genesNitrate Reductase1(NR1),NR2,Nitrate transporter 2.5(NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene,Super numeric nodules(SUNN). WhileMsSPL9–overexpressing transgenic plants have dramatically increased transcript levels ofSUNN,NR1,NR2, andNRT2.5, reducingMsSPL9caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of theMsSPL9transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest thatMsSPL9regulates nodulation in alfalfa in response to nitrate.