Flavonoids represent a large family of specialized metabolites involved in plant growth, development, and adaptation. Chalcone synthase (CHS) catalyzes the first step of flavonoid biosynthesis by directing carbon flux from general phenylpropanoid metabolism to flavonoid pathway. Despite extensive characterization of its function and transcriptional regulation, the molecular basis governing its posttranslational modification is enigmatic. Here, we report the discovery of a proteolytic regulator of CHS, namely, KFBCHS, a Kelch domain-containing F-box protein in Arabidopsis thaliana. KFBCHS physically interacts with CHS and specifically mediates its ubiquitination and degradation. KFBCHS exhibits developmental expression patterns in Arabidopsis leaves, stems, and siliques and strongly responds to the dark-to-light (or the light-to-dark) switch, the blue, red, and far-red light signals, and UV-B irradiation. Alteration of KFBCHS expression negatively correlates to the cellular concentration of CHS and the production of flavonoids. Our study suggests that KFBCHS serves as a crucial negative regulator, via mediating CHS degradation, coordinately controlling flavonoid biosynthesis in response to the developmental cues and environmental stimuli.