Methionine is the first limiting amino acid in maize-soybean meal diets for livestock and poultry. An imbalance in amino acids leads to inhibition of protein synthesis in the organism, thus affecting the quality of meat and milk. To analyse the regulatory mechanism of methionine accumulation in maize grains, this study used homologous cloning to obtain a candidate gene, ZmTS1 (Threonine Synthase 1), which affects methionine content.Methionine content in Zmts1 mutant seeds increased by 60%, SDS-PAGE results showed that the methionine residue-rich 10 KDa-δ alcohol-soluble protein in the mature Zmts1 mutant kernel was significantly increased compared to the wild type, confirming that the gene could significantly increase the methionine content in maize kernels.The gene contained a threonine synthase structural domain and the encoded protein was hydrophilic, according to biological information analysis. The gene contained a threonine synthase structural domain and the encoded protein was hydrophilic, according to biological information analysis. Transcriptome analysis revealed 1,144 differentially expressed genes related to methionine metabolism, of which 571 were up-regulated and 573 down-regulated. Analysis of GO and KEGG enriched pathways showed that the differentially expressed genes were mainly involved in amino acid biosynthesis and metabolism pathways. Seven key candidate genes involved in the methionine pathway were further validated by qRT-PCR, which could indirectly regulate the methionine pathway, and the expression patterns of RNA-Seq and qRT-PCR were consistent. This study provides new germplasm resources for breeding methionine-rich maize, and also provides some theoretical basis for the mechanism of methionine regulation in maize.