Striga is a major biotic constraint to sorghum production in semiarid tropical Africa and Asia. Genetic resistance to this parasitic weed is the most economically feasible control measure. Mutant alleles at the LGS1 (LOW GERMINATION STIMULANT 1) locus drastically reduce Striga germination stimulant activity. We provide evidence that the responsible gene at LGS1 codes for an enzyme annotated as a sulfotransferase and show that functional loss of this gene results in a change of the dominant strigolactone (SL) in root exudates from 5-deoxystrigol, a highly active Striga germination stimulant, to orobanchol, an SL with opposite stereochemistry. Orobanchol, although not previously reported in sorghum, functions in the multiple SL roles required for normal growth and environmental responsiveness but does not stimulate germination of Striga. This work describes the identification of a gene regulating Striga resistance and the underlying protective chemistry resulting from mutation.