AbstractWheat (Triticum aestivum) quality is mainly determined by grain storage protein compositions. Sulphur availability is essential for the biosynthesis of the main wheat storage proteins. In this study, the impact of different sulphur fertilizer regimes on a range of agronomically important traits and associated gene networks was studied. High-performance liquid chromatography was used to analyse the protein compositions of grains grown under four different sulphur treatments. Results revealed that sulphur supplementation had a significant effect on grain yield, harvest index, and storage protein compositions. Consequently, two comparative sulphur fertilizer treatments (0 and 30 kg ha⁻¹ sulphur, with 50 kg ha⁻¹ nitrogen) at seven days post-anthesis were selected for a transcriptomics analysis to screen for differentially expressed genes (DEGs) involved in the regulation of sulphur metabolic pathways. The International Wheat Genome Sequencing Consortium chromosome survey sequence was used as reference. Higher sulphur supply led to one up-regulated DEG and sixty-three down-regulated DEGs. Gene ontology enrichment showed that four down-regulated DEGs were significantly enriched in nitrogen metabolic pathway related annotation, three of which were annotated as glutamine synthetase. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment identified three significantly enriched pathways involved in nitrogen and amino acid metabolism.