〔Abstract〕 Objective To investigate the oxidative stress injury of nano zinc oxide nanoparticles(ZnO NPs) on human myocardial cells(AC16), and to analyze the mechanism of ZnO NPs from the transcriptome level. Methods Dynamic light scattering(DLS) was used to characterize and detect ZnO NPs. After AC16 cells were exposed to ZnO NPs at different doses and at different times, the cell survival rate was determined by CCK-8 method. AC16 cells were divided into control group, ZnO NPs(50, 100, 200 μmol/L), after 6 h treatment, the mitochondrial membrane potential(MMP) and reactive oxygen species(ROS) were measured. AC16 cells were divided into control group, 50 μmol/L ZnO NPs group and 200 μmol/L ZnO NPs group. After 6 h exposure, total RNA was extracted by TRIzol for transcriptome analysis, and the differentially expressed genes were enriched by gene body(GO), Kyoto Encyclopedia of Genes and Genomes(KEGG). Results The results of DLS showed that the hydrodynamic diameter was(192.2±1.63) nm and the Zeta potential was(-23.26±1.05) mV. CCK-8 results showed that the survival rate of AC16 cells decreased with the increase of dose and time of exposure to ZnO NPs. Fluorescence quantification showed that with the increase of ZnO NPs exposure dose, MMP significantly decreased at 100 μmol/L ZnO NPs(P<0.05), and ROS significantly increased at 50 μmol/L ZnO NPs(P<0.05).Using the multifunctional microplate reader, it was observed that MMP and ROS were statistically significant at 100 and 50 μmol/L ZnO NPs, respectively, showing a decrease in MMP and an increase in ROS. Transcriptome analysis showed that 1 071 genes were enriched in the 50 μmol/L ZnO NPs group compared with the control group, including 561 up-regulated genes and 510 down-regulated genes. Compared with the control group, 7 164 genes were enriched in 200 μmol/L ZnO NPs group, including 4 098 up-regulated genes and 3 066 down-regulated genes. GO and KEGG analysis showed that the differential genes were mainly concentrated in ROS, antioxidant activity, mitochondrial cytochrome C release, apoptosis and other signaling pathways. Conclusion ZnO NPs can decrease the survival rate of AC16 cells and induce mitochondrial damage and oxidative stress, among which ROS-mediated oxidative stress and mitochondrial function changes are important toxic mechanisms of ZnO NPs induced AC16 cytotoxicity.