Homology of medicine and food is the natural endowment of adlay crops, although it was always poor palatability and not easy to cook in food processing and utilization. Seed germination was the first step of the life cycle in plants, which could be an efficient process to improve palatability and quality properties. Aiming to provide theoretical insights for guidance in adlay functional food processing and industrial utilization, the metabolomics profiles of adlay seeds in four adlay varieties ‘Xingrenbaike’, ‘Qianyi 2 hao’, ‘Anguoyiyi’ and ‘Ribenyimi’ were measured under two treatments (before and after germination), followed by statistical analysis using widely-targeted metabolomics methods to identify the differential metabolites. Of 590 metabolites detected, 190 metabolites showing statistically different were identified. After germination, compounds of lipids, organic acids, alkaloids, lignans, coumarins, phenolic acids, amino acids and derivatives, nucleotide and derivatives were up-regulated in general, while flavonoids compounds were down-regulated. The top-ten ranking up-regulated metabolic molecules were N-Phenylacetylglycine, S-Adenosylmethionine, N-α-Acetyl-L-arginine, citraconic acid, alanylleucine, Asp-phe, L-theanine, Phe-Phe, O-p-coumaroyl-O-salicyloyl quinic acid, ribulose-5-phosphate, while the top-ten ranking down-regulated molecules were proline betaine, liquiritigenin-7,4-diglucoside, avicularin, cis-Zeatin, quercetin-3-O-α-L-arabinopyranoside, chrysoeriol-7-O-glucoside, peonidin, Kaempferide, esculin hydrate and raffinose. KEGG pathway enrichments showed that the metabolism pathways including amino acids biosynthesis, aminoacyl-tRNA biosynthesis, ABC transporters, pyrimidine metabolism, zeatin biosynthesis, flavonoid metabolism, alkaloid biosynthesis, glucosinolate biosynthesis and glyceride metabolism were changed significantly. However, some bioactive molecules with physiological functions were also found with significant enrichment, such as lysoPC(18:3), Coixol, 10-Formyl-THF, oleanolic acid, gallocatechin, chlorogenic acid, syringic acid, et. Several metabolic processes involved with biosynthesis of lipids, organic acids, alkaloids, lignans, coumarins, phenolic acids, amino acids and nucleotide and their derivatives, were robust active and enhanced during germination, and some characteristic metabolites and physiologically active molecules were hunted as well.