The existence of cancer stem cells (cScs) is considered to be the main reason for chemoresistance, metastasis and the ultimate failure of treatment in hepatocellular carcinoma (Hcc). However, there are a few chemical agents that may inhibit CSCs. The present study identified that 4,4'-bond secalonic acid D (4,4'-SAD), a compound isolated from the marine-derived fungus Penicillium oxalicum, inhibited the growth of side population (SP) cells isolated from human liver cancer cell lines Plc/PrF/5 and HuH-7 by attenuating the expression of aTP-binding cassette superfamily G member 2. Furthermore, the results of wound healing, Transwell, western blotting and reverse transcription-quantitative Pcr assays demonstrated that 4,4'-SAD suppressed the invasion and migration of SP cells by downregulating matrix metallopeptidase 9 (MMP-9) and upregulating the antagonist tissue inhibitor of metalloproteinases 1 in vitro. Moreover, in vivo study results found that 4,4'-SAD had anti-lung metastasis efficacy via the decrease of MMP-9 expression in the H22 Hcc model of Kunming mice. Therefore, the present study identified the potential of 4,4'-SAD as a promising candidate for the treatment of advanced liver cancer.