The population of CD133 positive cancer cells has been reported to be responsible for drug resistance of hepatocellular carcinoma (HCC). However, the potential molecular mechanism by which CD133+ HCC cells develop drug resistance is still unclear. In this study, we found that CD133+ HepG2 and Huh7 cells were resistant to cisplatin treatment, compared to the CD133- HepG2 and Huh7 cells. However, treatment with osthole, a natural coumarin isolated from umbelliferae plant monomers, was found to resensitize CD133+ HepG2 and Huh7 cells to cisplatin treatment. In the mechanism research, we found that treatment with osthole increased the expression of PTEN. As a result, osthole inhibited the phosphorylation of AKT and Bad to decrease the amount of free Bcl-2 in CD133+ HepG2 and Huh7 cells. Finally, cisplatin-induced mitochondrial apoptosis was expanded. In conclusion, combination treatment with osthole can resensitize CD133+ HCC cells to cisplatin treatment via the PTEN/AKT pathway.