Zinc (zeolitic) imidazolate framework 8 (ZIF-8) has been widely considered in the literature as an ideal candidate for drug delivery especially anti-cancer drugs. However, the available information on the biocompatibility and cytotoxicity of ZIF-8 nanoparticles is contradictory. Therefore, in the present study, the ZIF-8 particles were synthetized, characterized, and their potential toxicity on two eukaryotic cell lines including human embryonic kidney (HEK293) and human colon cancer (SW480) cells was investigated in vitro. The characterization of ZIF-8 particles by TEM, EDX, SEM, and DLS indicated the synthesis of the hexagonal crystals with mean diameter of 124.71±32.74 nm and the presence of the zinc element at 86.25% by weight (wt%) of the ZIF-8 structure. The results of the cytotoxicity assessment of ZIF-8 NPs showed that the viability of two different cell lines reduced significantly coincident with increasing exposure concentration from 0 to 500 μg mL−1 (P<0.05). The 24-h half-inhibitory concentration (IC50-24 h) values of ZIF-8 NPs for HEK293 and SW480 cell lines were 116.22 and 36.23 μg mL−1, respectively. We found that the viability of SW480 cells was significantly lower than the HEK293 cells in all exposure concentrations of ZIF-8 NPs except control. Exposure of both cells resulted in increasing of the intracellular reactive oxygen species (ROS) production and activation of apoptosis pathway. The apoptosis rate of cancer SW480 cells was higher than the normal HEK293 cells. These findings indicate that synthetized ZIF-8 NPs could be a candidate for cancer therapy, although their toxic effects on the normal cells also should be considered.