Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC.A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA orCompared with the corresponding adjacent tissues,the mRNA and protein levels ofClinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating