Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF^COI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF^COI1 complexes, suggesting a function for SCF^COI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF^COI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.