Abstract: To evaluate the cardiotoxicity of three novel proteasome inhibitors(NNU395, NNU458 and NNU459)in zebrafish, normal developmental zebrafish embryos at 6 hours post fertilization(hpf)were treated with different doses of NNU395 and NNU458 and NNU459 until 72 hpf, the zebrafish mortality was counted. Morphologic changes of the cardiovascular system were observed under a stereomicroscope, and the number of heart beats within 1 min was determined. The expression of cardiac development-related genes in zebrafish was detected by RT-qPCR(Quantitative Real-Time PCR). Results showed that NNU395, NNU458 and NNU459 increased the mortality of zebrafish in a concentration-dependent manner and the values of LC₅₀(50% lethal concentration)were(179. 7±12. 2), (27. 5±1. 3)and(24. 4±2. 6)μmol/L, respectively. Moreover, the toxicity of our three compounds in zebrafish are less when compared with their modified precursors. Upon administration of NNU395 at the concentrations of 120- 200 μmol/L, and NNU458 or NNU459 at the concentration of 30 μmol/L, the zebrafish showed obvious pericardial edema cardiac malformation. 120- 200 μmol/L NNU395 and 0. 1- 30 μmol/L NNU458 or 10- 30 μmol/L NNU459 significantly reduced the heart rate of zebrafish. All of three compounds at the tested concentration had no significant effects on the expression of the heart development-related genes in zebrafish. Our results suggested that low concentrations of NNU395, NNU458 and NNU459 have no obvious toxicity on cardiac development of zebrafish. While, higher concentrations of them showed cardiovascular toxicity on zebrafish.