Background: Soybean [Glycine max (L.) Merrill] oil is a complex mixture of five fatty acids (oleic, linoleic, linolenic, palmitic and stearic). As a vegetable oils, soybean oil has a less than desirable fatty acid composition. The high content of linoleic acid contributes to poor oil oxidative stability. Soybean oil with lower linoleic acid content is desirable. To investigate the genetic architecture of linoleic acid in soybean seed, 510 soybean germplasms from China were collected as natural populations. Results: Phenotypic identification results showed that the content of linoleic acid varied from 36.22% to 72.18%. After a total of 2,423,512 nucleotide polymorphism (SNP) were obtained, a new candidate gene Glyma.04G116500.1 (GmWRI14) related to linoleic acid was discovered by 3-year long Genome-wide association analysis (GWAS). GmWRI14 belongs to the plant WRI1 protein family. The GmWRI14 showed a negative correlation with the linoleic acid content and the correlation coefficient was -0.912. To test whether GmWRI14 can lead to lower linoleic acid content in soybean seed, we introduced GmWRI14 into the soybean genome. Overexpression of GmWRI14 leads to higher accumulation of oil with lower linoleic acid content in soybean seed. RNA-seq verified that GmWRI14-overexpressed lines showed lower accumulation of GmFAD2-1 and GmFAD2-2b than non-transgenic lines. Conclusions: These results indicate that the down-regulation of FAD2 gene triggered by the transcription factor WRI1 is the underlying mechanism reducing linoleic acid level of seed oil. GmWRI14 is a new key candidate gene related to linoleic acid. These results will significantly improve understanding of the transcriptional control exerted by GmWRI14.