Costunolide (COST) is a sesquiterpene lactone that belongs to the germacranolide group, and occurs mainly in Saussurea lappa Clarke. Although COST inhibits the proliferation and metastasis of cancer cells and induces their apoptosis, it suffers poor water solubility and cellular permeability. Therefore, this study aimed to enhance the anti-proliferative activity of COST in LS174T colon cancer cells through its inclusion in bilosomal nanoformulation (COST-BILs). The optimized BIL formula contained cholesterol and Span-85 in a molar ratio of 1:5 as well as bile salt at a molar concentration of 0.5 mM, with entrapment efficiency of 63.4 ± 3.59 % and particle size of 119.7 ± 3.63 nm. The optimized COST-BILs showed a potent cytotoxic effect against LS174T cells with an IC₅₀ of 6.20 µM; meanwhile, raw COST had an IC₅₀ of 15.78 µM. Safety and relative selectivity were confirmed in the normal human colonic epithelial cells (HCoEpC). Cell cycle analysis indicated that both raw COST and COST-BILs significantly increased the fraction of LS174T cells in the sub-G1 phase. This was accompanied by a significant enhancement of early, late, and total apoptosis, as indicated by annexin-V staining. In addition, COST-BILs exhibited more potent activity in up-regulating CASP3, TP53, and BAX, and in down-regulating the expression of BCL2 mRNA as compared to raw COST. Further, the prepared formula enhanced the release of cytochrome C as well as the generation of reactive oxygen species (ROS) and reduced the integrity of mitochondrial membranes. In conclusion, the loading of COST on BILs significantly enhances its pro-apoptotic activity in LS174T cells.