Fatty acid (FA) levels and profiles are vital for soybean oil quality, while cytokinins (CKs) and abscisic acid (ABA) are potent regulators of plant growth and development. Previous research suggested associations between FA biosynthesis and hormonal signalling networks; however, hormonal regulation of FA accumulation during soybean (Glycine max) seed maturation has never been measured. We analysed hormone and FA profiles obtained from HPLC-(ESI)-MS/MS and GC-FID screening during soybean seed maturation. A multilayered data processing approach, involving heat-maps, principal component analysis (PCA), correlation and multiregression models, suggested a strong relationship between hormone metabolism and FA/oil accumulation during seed maturation. Most strikingly, positive correlations were found between the levels of CK ribosides [transZeatin riboside (tZR), N6-isopentenyladenosine (iPR)] at the early stages of SM (R5-R6) and C18:0, C18:2 and oil content at the R8 stage. Moreover, multiple regression models revealed functional linkages between several CK derivatives and FA and oil content in mature seeds. To further test the significance of hormone regulation in FA metabolism, plants of two soybean accessions with contrasting hormone and FA profiles were sprayed with exogenous ABA and transZeatin (tZ) during the seed-filling period (R5-R6). Depending on the hormone type and concentration, these treatments distinctly modified biosynthesis of all tested FAs, except for C18:0. Most remarkably, tZ (50 nM) promoted production of C16:0, C18:1, C18:2, C18:3, and oil accumulation in maturing seeds. Overall, the results indicate impactful roles for ABA and CKs in FA accumulation during SM and represent a further step towards understanding FA biosynthesis, and potential improvements of soybean oil profiles.