In this study, pillar[5]arene containing ten terminal alkynyl-functional group was prepared and carried out an optimization procedure for target molecule with using 5-hydroxyquinoline molecule known with biological activity. The novel molecule was named as iso-QP[5] and characterized by FT-IR, melting point, NMR, elemental analysis, and mass spectroscopy. The aim was to evaluate the anticancer effects of iso-QP[5] on MCF7 human breast cancer cells. The IC50 dose of iso-QP[5] in MCF7 cells was found to be 25 µM for 24 h using XTT assay. Expressions of eight genes for apoptosis were determined by qPCR. The results showed a significant increase in the expression of BAX, CASP3, CASP9, CYCS, and P53 genes after treatment with iso-QP[5] in MCF7 cells. However, iso-QP[5] treatment significantly decreased expression of BCL2 and CASP8 genes. It is concluded that novel molecule iso-QP[5] caused apoptosis by regulating genes that are important in apoptosis in human breast cancer cells.