CITRON (rho-interacting, serine/threonine kinase 21), which is a key component of the midbody, is essential for cytokinesis. However, the role of CITRON in hepatocellular carcinoma (HCC) is poorly understood. Here we first measured the expression of CITRON in HCC specimens by quantitative real-time RT–PCR and immunohistochemical staining. The results showed CITRON to be frequently up-regulated in HCC as compared with adjacent non-tumour tissues. Then we employed adenovirus-mediated RNA interference against CITRON to assess its anti-proliferation effect on SMMC-7721 cells, a representative HCC cell line. The resulting data demonstrated that CITRON knockdown was capable of inhibiting the proliferation and colony formation of SMMC-7721 cells, with an obvious increase of multinucleated cells. Furthermore, we subcutaneously injected the SMMC-7721 cells with the CITRON knockdown into nude mice to evaluate the tumourigenicity. The data indicated that adenovirus-mediated RNA interference can suppress tumourigenicity in vivo of HCC cells. Our data suggest that CITRON may be a potential target for therapeutic intervention in HCC.