During the last years, special interest has been focused on the recovery of bioactive compounds from food industry wastes in an effort to find added value applications for residues. In this work, Opuntia spp. residues from fruit juice production were explored as potential sources of natural chemotherapeutic ingredients towards colon cancer. Hydroalcoholic extraction and adsorption separation processes (with Amberlite XAD16 resin) were used to produce natural extracts from residues of Opuntia ficus-indica and Opuntia robusta and their antiproliferative effect was evaluated in human colon carcinoma HT29 cell line. Our results showed that the natural extracts efficiently inhibited cancer cell growth and induced cell cycle arrest in different checkpoints-G1, G2/M and S. The phytochemical constituents presented in the samples, namely betacyanins, flavonoids (isorhamnetin derivatives) and phenolic acids (ferulic acid) were identified as the main responsible compounds for the cell cycle arrest. Moreover, the extracts that presented the lowest effective dose values in terms of polyphenols (ED50 value < 0.5 mg GAE/mL) showed to increase intracellular ROS accumulation in HT29 cells, suggesting that cancer cell death may be induced by the pro-oxidant effect of these compounds. (C) 2013 Elsevier Ltd. All rights reserved.