The toxic effect of TiO2 nanoparticles (TNP) greatly varies with the variation in synthesis protocol followed. Any morphological alteration of TNPs affects their activity. In the present study, we report the detailed toxicological analysis of TNPs fabricated by a microwave irradiation–assisted hybrid chemical approach. The toxicological mechanism was studied in human colon cancer cell lines (HCT116). Results indicate that TNP induces oxidative stress on HCT116, which, in turn, causes mitochondrial membrane depolarization. We also observed activation of Bcl-2 and caspase-3 by Western blot analysis. This indicates TNPs induce mitochondrial-mediated apoptosis. Furthermore, G1 cell cycle arrest was observed by flow-cytometric analysis. This study provides an understanding of the mechanism of action for apoptosis induced by TNPs, which can be further used to design safe TNPs for various consumer products and also suggests that extensive research needs to be done on harmful effects of TNPs synthesized from different approaches before commercial application. © Copyright © 2020 Ranjan, Dasgupta, Mishra and Ramalingam.