The 9-fatty acid desaturase introduces a double bond at the 9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how 9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole 9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a di-proline motif, which plays a crucial role in the regulation of 9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.