Background: Rho-Associated kinases ROCK1 and ROCK2 have been extensively investigated in the pathogenesis of cardiovascular disease. However, their roles are not fully understood in carcinogenesis. In this study, we investigated whether ROCK1 or ROCK2 is required for the survival and growth of hepatocellular carcinoma (HCC) cells and underlying mechanism. Methods: ROCKs expression was determined in human HCC tissue and cell lines using qRT-PCR, western blotting, and immunohistochemistry (IHC). Cell growth and proliferation were assayed using cell counting kit-8 (CCK-8) and EdU incorporation assay. Cell cycle and apoptosis analysis were performed using flow cytometry. HCC cell division or mitosis was observed using a confocal microscope and a time relapse fluorescence microscope. Inhibitory role of targeting ROCK1/2 on HCC was assayed in both xenograft and primary HCC mouse models. Results: Both ROCK1 and ROCK2 are over-expressed in human HCC tissues and cell lines. Knockdown of ROCK1 or ROCK2 inhibited HCC cell growth. Pharmacological inactivation of ROCK1/2 with Fasudil further blocked the growth and survival of HCC both in vitro and in vivo. Mechanically, Fasudil induces cell cycle arrest in HCC cells, but not apoptosis. Instead, Fasudil treatment led to mitotic catastrophe in HCC cells, characterized with the multipolar and asymmetric mitosis, and disassociated stress fibers. Knockdown of cofilin restored the cell morphology and division, and reduced the mitotic catastrophe induced by Fasudil. Conclusions: Both ROCK1 and ROCK2 are required for HCC cell division and growth. Targeting ROCK1 or ROCK2 rather than both can serve as a potential approach for HCC treatment and may reduce the side effects.