The silkworm cocoon colour has attracted researchers involved in genetics, physiology and ecology for a long time. 'Ryokuken' cocoons are yellowish green in colour due to unusual flavonoids, prolinylflavonols, while 'Sasamayu' cocoons are light green and contain only simple flavonol glucosides. We found a novel gene associated with the cocoon colour change resulting from a change in flavonoid composition and named it Lg (light green cocoon). In the middle silk glands of the + Lg /+ Lg larvae, 1-pyrroline-5-carboxylic acid (P5C) was found to accumulate due to a decrease in the activity of pyrroline-5-carboxylate reductase (P5CR), an enzyme reducing P5C to proline. Sequence analysis of BmP5CR1, the candidate gene for Lg, revealed a 1.9 kb insertion and a 4 bp deletion within the 1st intron, a 97 bp deletion within the 4th intron, and a > 300 bp insertion within the 3'-UTR, in addition to two amino acid changes on exons 3 and 4 in + Lg /+ Lg compared to Lg/Lg. Decreased expression of BmP5CR1 was observed in all of the investigated tissues, including the middle silk glands in + Lg /+ Lg , which was probably caused by structural changes in the intronic regions of BmP5CR1. Furthermore, a BmP5CR1 knockout strain exhibited a yellowish green cocoon with the formation of prolinylflavonols. These results indicate that the yellowish green cocoon is produced by a BmP5CR1 deficiency. To our knowledge, this is the first report showing that the defect of an enzyme associated with intermediate metabolism promotes the conjugation of phytochemicals derived from foods with endogenously accumulating metabolites in animal tissues.