Cathepsin L protease, which belongs to the papain-like cysteine proteases family, is an important player in many physiological and pathological processes. However, little was known about the role of cathepsin L in ladybird beetles (Coccinella septempuctata Linnaeus) during diapause. Here, we analyzed the characteristics of cathepsin L (CsCatL) in the females of C. septempunctata and its role during the diapause of the ladybeetle. CsCatL was cloned and identified from beetle specimens by rapid amplification of cDNA-ends (RACE). The cDNA sequence of CsCatL was 971 bp in length, including an 843 bp open reading frame encoding a protein of 280 amino acids. It was identified as the cathepsin L group by phylogenetic analysis. Knockdown of CsCatL by RNA interference led to decreased expression levels of fatty acid synthase 2 (fas 2) genes and suppressed lipid accumulation. Furthermore, silencing the CsCatL gene distinctly reduced diapause-related features and the survival of female C. spetempunctata under diapause-inducing conditions. The results suggested that the CsCatL gene was involved in fatty acid biosynthesis and played a crucial role in the survival of adult C. septempunctata during the diapause preparation stage.