Nanoparticles have garnered considerable scientific attention in recent years due to their diagnostic and therapeutic applications in cancer. The purpose of this study was to determine the effect of superparamagnetic iron oxide nanoparticles (FeO MNPs) on the induction of apoptosis in human colorectal adenocarcinoma cell line (HT-29) cells. The purpose of this study was to elucidate the mechanisms of apoptosis induced by FeO MNPs following MTT assay and to determine the optimal dose of 2.5 g/mL for inducing apoptosis in HT-29 cells. In HT-29 cells, FeO MNPs increased reactive oxygen species (ROS), calcium ion (Ca), and DNA damage. Additionally, the FeO MNPs significantly increased caspase 3 and 9 expression and decreased Bcl-2 expression at the protein and mRNA levels when compared to the control group (P = 0.0001). FeO MNPs also induced apoptosis in cancer cells by increasing the level of (ROS) and intracellular Ca, followed by an increase in caspase 3 and 9 expression and a decrease in Bcl-2 expression and direct DNA damage. FeO MNPs are an appropriate choice for colon cancer treatment based on their cell toxicity and induction of apoptosis in HT29 cells.