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This work, investigated the effect of selenium (Se) on foxtail millet. The results showed that foliar sodium selenite (Na 2 SeO 3) applied at critical growth stages significantly improved the Se content of millet. The application of 120 g hm−2 Na 2 SeO 3 led to a 9.8-fold increase in selenomethionine (SeMet) and selenocysteine (SeCys 2) in the millet. While Se treatment elevated the K and Fe content in the millet. Proteomic analysis using relative quantification was performed on millet during the harvest stage. A comparison between Se-enriched millet and the control group, identified 123 differentially expressed proteins. The proteins involved in carbohydrate and amino acid metabolism were the most profoundly affected by Se treatment. The results provided necessary information about the Se response in foxtail millet at the proteome level, which could be useful for analyzing the Se response pathway and producing Se-enriched foxtail millet in the future. Image 1 • SeMet and SeCys 2 were detected in foxtail millet. • Foliar spraying of selenite increased Se content (SeMet + SeCys 2) in foxtail millet. • Se treatment affected K and Fe content in foxtail millet. • 123 differentially expressed proteins were identified by proteomic. • Carbohydrate metabolism and amino acids metabolism were affected by Se treatment. [ABSTRACT FROM AUTHOR]