Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of theArabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Twochil mutants with a seed colour similar to that of wild-type Arabidopsishave been identified here, but in sharp contrast to the characteristictransparent testa seed phenotype associated with other known flavonoidpathway genes. CHIL loss-of-function mutations led to a strong reductionin the proanthocyanidin and flavonol levels in seeds, but not in theanthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, andinteracts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer inthe flavonoid pathway.