Parthenolide (PT), a sesquiterpene lactone extracted from the plant feverfew, has been demonstrated to have anti-inflammatory and anticancer properties. Although PT has been revealed to markedly inhibit colorectal cancer cell proliferation, the inhibitory effects decrease with administration time. These findings revealed that colorectal cancer cells develop resistance to PT. However, the underlying mechanism is unclear. In the present study we observed significantly low expression of Smad4 in 3 PT-resistant cell lines (HCT‑116/PT, HT-29/PT and Caco-2/PT), which were obtained using in vitro concentration gradient-increased induction, but not in their parental cells. In the present study we used the lentiviral‑mediated transfection method to upregulate Smad4 in resistant colorectal cancer cell lines. Flow cytometry assay was used to assess cell apoptosis. Cell migration was detected using a QCM™ 24-well Fluorimetric Cell Migration Assay kit. Our study showed that Smad4 overexpression notably decreased the half maximal inhibitory concentration (IC50) values for PT in the 3 PT-resistant cell lines, and improved the inhibitory effects of PT on cell migration and enhanced apoptosis in vitro as well as suppressed xenografted tumors in a PT-resistant colorectal cancer mouse model. Further study by western blotting into the underlying mechanism demonstrated that Smad4 overexpression suppressed the expression of MDR1 in the resistant cells, and resulted in the accumulation of PT, which in turn promoted the expession of caspase-3 and Bax and inhibited the expression of Bcl-2 and the phosphorylation of NF-κB p65. In short, Smad4 re-expression may be crucial for enhancing the sensitivity and reversing the resistance to PT in PT-resistant colorectal cancer cells.