With the current worldwide increasing use of plastics year by year, nanoplastics (NPs) have become a global threat to environmental and public health concerns. Among plastics, polypropylene (PP) is widely used in industrial and medical applications. Owing to the lack of validated detection methods and standard materials for PP NPs, understanding the impact of PP NPs on the environmental and biological systems is still limited. Here, isotactic polypropylene (iPP) was fabricated into oxidized polypropylene micro/nanoplastics (OPPs) via a thermal oxidation using hydrogen peroxide (H2O2) under various heating temperatures. The resulting OPPs were investigated in terms of the size distribution, surface chemistry, morphology, and thermal property as well as their concentration-dependent cytotoxicity to a human intestinal epithelial cell line (Caco-2), which could be a route to uptake NPs into the body through the food chain. The average diameters of the OPPs decrease with increasing reaction temperature. The OPPs obtained at 175 °C (OPP175) were spherical in shape and had a rough surface, with size distributions of approximately 0.14 ± 0.02 μm. A significant increase in the carbonyl content of the oxidized product was confirmed by Fourier transform infrared and X-ray photoelectron spectroscopy analyses. Caco-2 cells were exposed to OPP175 in a dose-dependent manner, and a significant loss of cell viability occurred at the concentration of 100 μg/mL. Thus, this study provides a fundamental approach for the fabrication of a model of NPs for the urgently demanded in vitro and in vivo studies to assess the potential impact of NPs on biological systems.