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Protein content (PC) is crucial to the nutritional quality of soybean [Glycine max (L.) Merrill]. In this study, a total of 266 accessions were used to perform a genome-wide association study (GWAS) in three tested environments. A total of 23,131 high-quality SNP markers (MAF ≥ 0.02, missing data ≤ 10%) were identified. A total of 40 association signals were significantly associated with PC. Among them, five novel quantitative trait nucleotides (QTNs) were discovered, and another 32 QTNs were found to be overlapping with the genomic regions of known quantitative trait loci (QTL) related to soybean PC. Combined with GWAS, metabolome and transcriptome sequencing, 59 differentially expressed genes (DEGs) that might control the change in protein content were identified. Meantime, four commonly upregulated differentially abundant metabolites (DAMs) and 29 commonly downregulated DAMs were found. Remarkably, the soybean gene Glyma.08G136900, which is homologous with Arabidopsis hydroxyproline-rich glycoproteins (HRGPs), may play an important role in improving the PC. Additionally, Glyma.08G136900 was divided into two main haplotype in the tested accessions. The PC of haplotype 1 was significantly lower than that of haplotype 2. The results of this study provided insights into the genetic mechanisms regulating protein content in soybean.