To study the role of heat shock protein 90 (HSP90) in the development of zebrafish embryos, we examined the mRNA levels of two HSP90 activity marker genes, namely (heat shock protein beta-1), at different developmental stages. Furthermore, we observed the marker gene expression and development of zebrafish embryos at different stages after treating with 2 μmol/L, 5 μmol/L, and 10 μmol/L of the HSP90 inhibitor radicicol. Analysis by quantitative polymerase chain reaction showed a strong increase in the mRNA levels of when zebrafish embryos were treated with radicicol at 12 or 24 hours post-fertilization (hpf). Western blot results showed that the protein level of HSP70 was upregulated when zebrafish embryos were treated with 5 μmol/L radicicol at 24 hpf, indicating that the activity of HSP90 was inhibited. Observations of embryonic development showed that the radicicol-treated embryos had a delayed development and survival rates of 95%, 77%, and 35% after treatment with 2 μmol/L, 5 μmol/L, and 10 μmol/L radicicol, respectively. The embryos manifested some abnormalities including reduced pigmentation, an enlarged pericardial membrane, and muscular dystrophy after treatment with 5 μmol/L radicicol at 72 hpf. The results showed that HSP90 plays an important role on the development of zebrafish embryos. The radicicol concentration of 5 μmol/L appeared optimal, because it strongly inhibited HSP90 without decreasing the survival rate of zebrafish embryos below 75%. Various morphological changes appeared in the radicicol-treated embryos, laying a foundation for the further study of HSP90's functions in zebrafish embryos.