Abstract: Ultrasonic treatment has been widely used to modulate the metabolite profiles in germinated sorghum. This study aims to elucidate the molecular mechanisms after ultrasonic treatment using integrated metabolomic and transcriptomic analysis. Sorghum seeds were divided into three groups: G0 (ungerminated control group), G48 (germinated for 48 h), and GUT48 (ultrasonically assisted germination for 48 h). The widely targeted metabolomics analysis was conducted using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Furthermore, the 1,014 metabolites were identified, which were dominated by amino acids and their derivatives (17.97%), followed by flavonoids (17.08%), lipids (11.15%), carbohydrates (9.77%), and organic acids (8.79%). Differentially accumulated metabolites (DAMs) were defined as VIP > 1.0, |log₂FC| ≥ 0.585 (FC > 1.5 or < 0.667), and P < 0.05 between two groups (G0 vs G48 and G0 vs GUT48). Compared with the G0, 265 DAMs (56 up- and 209 down-regulated) were detected in the G48, whereas 339 DAMs were found in GUT48; Among them, 268 DAMs (212 up- and 56 down-regulated) specifically responded to the ultrasound, which were enriched mainly in amino acid metabolism, carbohydrate metabolism, and secondary metabolite biosynthesis. The results indicated that the ultrasonic treatment induced a more significant metabolic reprogramming compared with the natural germination. The ultrasound acted as an effective physical stimulus, in order to enhance the biosynthetic pathways and metabolic fluxes in the germinated sorghum. Transcriptomic analysis was conducted to further explore the transcriptional regulation underlying these metabolic variations. The DESeq2 (Differential Expression analysis for Sequence data 2) package was utilized to identify a total of 35,047 genes. Differential expression genes (DEGs) screening criteria were set as |log₂FC| ≥ 1 (FC > 2 or < 0.5) and padj < 0.05. A total of 13604 DEGs were identified between G0 and GUT48, including 1,841 ultrasound-responsive DEGs (1097 up- and 744 down-regulated). Gene Ontology (GO) enrichment analysis revealed that these DEGs were significantly associated with catalytic activity, oxidoreductase activity, and transmembrane transport. While Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis shared the enrichment in the carbohydrate metabolism, amino acid biosynthesis, and flavonoid biosynthetic pathways. The integrated metabolomic and transcriptomic analysis further revealed that the ultrasonic treatment significantly upregulated the expression of the genes encoding key enzymes, such as hexokinase (HXK), fructokinase (FRK), and pyruvate kinase (PK), which played the central roles in glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. The enhanced expression of these genes promoted the accumulation of the metabolic intermediates, such as glucose-6-phosphate and α-ketoglutarate. Both energy production and the biosynthesis of the carbon skeletons were facilitated for the secondary metabolism. In amino acid metabolism, the expression of genes encoding enzymes was upregulated significantly, such as glutamate decarboxylase (GAD) and various aminotransferases, leading to the elevated levels of essential amino acids, including leucine, phenylalanine, and their biosynthetic precursors. Moreover, ultrasound treatment enhanced the expression of the genes related to the phenylpropanoid and flavonoid biosynthesis pathways, such as phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), resulting in the higher accumulation of bioactive flavonoids, including apigenin, ferulic acid, and coumaric acid. Moreover, the qRT-PCR was conducted on 5 ultrasonic-responsive DEGs (LOC8074774, LOC8066166, LOC8059757, LOC8062170, LOC8077078), in order to verify transcriptome data. Five genes showed the lowest expression in G0, moderate increase in G48, and significant upregulation in GUT48. All trends were consistent with the transcriptome data. The ultrasound promoted the conversion of the primary metabolites into secondary metabolites, thus enhancing the nutritional and functional value of germinated sorghum. Overall, a molecular framework was provided to link the ultrasonic stimulation to the regulation of the key metabolic networks in germinated sorghum. The metabolomic and transcriptomic data revealed that the ultrasound-assisted germination accelerated the seed metabolism, while the biomass accumulation enriched the essential nutrients and bioactive compounds. Ultrasonic technology can serve as a sustainable and non-thermal processing to enhance the nutritional and functional properties of the value-added sorghum functional products. The finding can provide some insights into the metabolic plasticity of the sorghum under ultrasound-assisted germination.