A series of twenty seven oxa/azaspiro[4,5]trienone derivatives were synthesized and their anticancer properties have been explored. GI50 values of all these compounds were evaluated against four types of human cancer cell lines, i.e. MCF-7 (breast), DU-145 (prostate), A549 (lung) and HepG2 (liver). Five compounds of the series exhibited good anticancer potential against MCF-7 with GI50 values less than 2 μM. Detailed biological studies of the two representative compounds 9b and 9e revealed that they arrest cell cycle in G0/G1 phase and induce mitochondria mediated apoptosis, that was further confirmed by measurement of mitochondrial membrane potential (ΔΨm), intracellular ROS generation, caspase 9 activity and Annexin V-FITC assay. Furthermore, western blot analysis suggested that these compounds up-regulate the levels of p53, p21, p27 and Bax, and down-regulate the level of Bcl-2 confirming the apoptosis inducing properties.