Objective To investigate the regulatory effect of citron rho-interacting serine/threonine kinase (CIT) on drug resistance of adriamycin (ADM) resistant hepatocellular carcinoma cells and its mechanism.   Methods Random nonsense negative sequence (si-NC), CIT small interfering RNA (si-CIT) trasfected into SK-Hep1/ADM cells with liposomes. Real-time PCR, Western blotting were used to detect the expression of CIT, X linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma protein 2 (Bcl-2), Bcl-2 related X gene (Bax), cytochrome C (Cyt C), phosphatase gene induction putative kinase 1 (PINK1), autosomal recessive adolescent Parkinson’s disease pathogenic gene (Parkin) and autophagy microtubule-associated protein light chain 3 antibody Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ); Cell counting kit 8(CCK-8), flow cytometry, and fluorescence probe(JC-10) staining were used to detect cell inhibition rate, apoptosis and mitochondrial membrane potential.   Results The expression level of CIT in drug-resistant cells was significantly higher than that in normal cells. Knocking down CIT significantly increased the sensitivity to ADM. Knocking down CIT promoted drug-resistant cell apoptosis and mitochondrial autophagy, down-regulated cell mitochondrial membrane potential, and up-regulated Bax, Cyt C, and PINK1, Parkin,LC3-Ⅰ/Ⅱ, down-regulate XIAP, Bcl-2, Cyt C.   Conclusion CIT gene could regulate the drug resistance of adriamycin  resistant HCC cells through mitochondrial pathway apoptosis and autophagy.