Summary:Soybean cyst nematode (SCN, Heterodera glycines) is a major pathogen harmful to soybean all over the world, causing huge yield loss every year. Soybean resistance to SCN is a complex quantitative trait controlled by a small number of major genes (rhg1 and Rhg4) and multiple micro‐effect genes. Therefore, the continuous identification of new resistant lines and genes is needed for the sustainable development of global soybean production. Here, a novel disease‐resistance quantitative trait locus Rscn‐16 was identified and fine mapped to an 8.4‐kb interval on chromosome 16 using an F2 population. According to transcriptome and metabolome analysis, a UDP‐glucosyltransferase encoding gene, GmUGT88A1, was identified as the most likely gene of Rscn‐16. Soybean lines overexpressing GmUGT88A1 exhibited increased resistance to SCN, higher isoflavone glycosides and larger seed size while the phenotype of RNA‐interference and knockout soybean lines showed sensitivity to SCN and decreased in seed size compared to wild‐type plants. GmMYB29 gene could bind to the promoter of GmUGT88A1 and coordinate with GmUGT88A1 to regulate soybean resistance to SCN and isoflavone accumulation. Under SCN infection, GmUGT88A1 participated in the reorientation of isoflavone biosynthetic metabolic flow and the accumulation of isoflavone glycosides, thus protecting soybean from SCN stress. GmUGT88A1 was found to control soybean seed size by affecting transcription abundance of GmSWEET10b and GmFAD3C, which are known to control soybean seed weight. Our findings provide insights into the regulation of SCN resistance, isoflavone content and seed size through metabolic flux redirection, and offer a potential means for soybean improvement.