A new type of azomethine ylides, which was in situ generated by the reaction of ethyl glycinate hydrochloride and dimethyl acetylenedicarboxylate, reacted with 3-phenacylideneoxindoline-2-ones in ethanol to give polysubstituted spiro[indoline-3,3′-pyrrolidines] in good yields. The biological activities of the various spiro[indoline-3,3′-pyrrolidines] obtained were investigated by in vitro evaluation against mouse breast cancer cells 4T1, mouse colon cancer cells CT26, human liver cancer cells HepG2 and human lung cancer cells A549 by MTT assay. The results demonstrated that eight compounds showed cytotoxicity towards HepG2 cells, and compound 2j showed promising cytotoxicity towards CT26 cells with an IC₅₀ value of about 50 μg mL⁻¹ in 24 h.