Understanding the biocompatibility of nanoparticles in dental materials is essential for their safe usage in the oral cavity. In this study, we investigated whether nanoparticles deposited on orthodontic latex rubber bands are involved in the induction of cytotoxicity. A method of stretching to three times (“3L”) the length of the latex rubber bands was employed to detach the particles using the original length (“L”) for comparison. The cytotoxicity tests were performed on extracts with mouse fibroblasts (L929) and human gingival fibroblasts (HGFs). Fourier transform infrared spectroscopy, ion chromatography, elemental analysis, and inductively coupled plasma mass spectrometry (ICP-MS) were performed to detect the harmful components in the extracts from rubber bands. There was a significant decrease in the cell viability in the “L” samples compared with the “3L” samples (P<0.05) in the L929 and HGF cells. This was due to the Ni single crystal nanoparticles (~50nm) from the inner surface of “L” samples that were detached in the “3L” samples as well as the Zn ion (~9 ppm) detected in the extract. This study revealed that the Ni nanoparticles, as well as Zn ions, were involved in the induction of cytotoxicity from the latex rubber bands.