Seeds of root parasitic plants, Striga, Orobanche and Phelipanche spp., are induced to germinate by strigolactones exudated from host roots. In Striga-resistance cultivars of Sorghum bicolor, the loss-of-function of the Low Germination Stimulant 1 (LGS1) gene changes the major strigolactone from 5-deoxystrigol to orobanchol having the opposite C-ring stereochemistry. However, the biosynthetic pathway of 5-deoxystrigol catalyzed by LGS1 has not been fully elucidated. Since the other unknown regulator in addition to LGS1 encoding a sulfotransferase appeared to be necessary for the stereoselective biosynthesis of 5-deoxystrigol, we examined Sobic.005G213500 (Sb3500) encoding a 2-oxoglutarate-dependent dioxygenase as a candidate, which is co-expressed with LGS1 and located on the 5'-upstream of LGS1 in the sorghum genome. When LGS1 was expressed with known strigolactone biosynthetic enzyme genes including the cytochrome P450 SbMAX1a but not Sb3500 in Nicotiana benthamiana leaves, 5-deoxystrigol and its diastereomer 4-deoxyorobanchol were produced in approximately equal amounts, while the production of 5-deoxystrigol was significantly larger than that of 4-deoxyorobanchol when Sb3500 was co-expressed in addition to them. We also confirmed the stereoselective 5-deoxystrigol production by an in vitro feeding experiment using synthetic chemicals with recombinant proteins expressed in E. coli and yeast. This finding demonstrated that Sb3500 is a stereoselective regulator in the conversion of the strigolactone precursor carlactone to 5-deoxystrigol catalyzed by LGS1 and SbMAX1a, providing a detailed understanding of how different strigolactones are produced to combat parasitic weed infestations.