Hepatocellular carcinoma (HCC) has a relatively higher incidence in many countries of Asia. Globally, HCChas a high fatality rate and short survival. Epirubicin, a doxorubicin analogue, may be administered alone or incombination with other agents to treat primary liver cancer and metastatic diseases. However, the toxic effects ofepirubicin to normal tissues and cells have been one of the major obstacles to successful cancer chemotherapy.Here, we investigated the effects of epirubicin in combination with kappa-selenocarrageenan on mice with H22implanted tumors and HepG-2 cell proliferation, immune organ index, morphology, cell cycle and related proteinexpressions in vivo and in vitro with sequential drug exposure. The inhibitory rate of tumor growth in vivo wascalculated. Drug sensitivity was measured by MTT assay, and the King’s principle was used to evaluate theinteraction of drug combination. Morphological changes were observed by fluorescent microscopy. Cell cyclechanges were analyzed by flow cytometry. Expression of cyclin A, Cdc25A and Cdk2 were detected by Westernblotting. In vivo results demonstrated that the inhibitory rate of EPI combined with KSC was higher than thatof KSC or EPI alone, and the Q value indicated an additive effect. In addition, KSC could significantly raise thethymus and spleen indices of mice with H22 implanted tumors. In the drug sensitivity assay in vitro, exposure toKSC and EPI simultaneously was more effective than exposure sequentially in HepG-2 cells, while exposure toKSC prior to EPI was more effective than exposure to EPI prior to KSC. Q values showed an additive effect inthe simultaneous group and antagonistic effects in the sequential groups. Morphological analysis showed similarresults to the drug sensitivity assay. Cell cycle analysis revealed that exposure to KSC or EPI alone arrested thecells in S phase in HepG-2 cells, exposure to KSC and EPI simultaneously caused accumulation in the S phase,an effect caused by either KSC or EPI. Expression of cyclin A, Cdc25A and Cdk2 protein was down-regulatedfollowing exposure to KSC and EPI alone or in combination, exposure to KSC and EPI simultaneously resultingin the lowest values. Taken together, our findings suggest that KSC in combination with EPI might have potentialas a new therapeutic regimen against HCC.