<p>Hexavalent chromium [Cr(VI)] and hexavalent uranium [U(VI)] are common heavy metal pollutants in aquatic environments, which are harmful to aquatic organisms. As a keystone species representing reptiles in freshwater ecosystems, Chinese soft-shelled turtles serve as sentinel species in environmental monitoring. However, the toxic effects of these heavy metal pollutants on this species have not been fully elucidated. This study investigated the toxic effects, and underlying mechanisms of Cr(VI) and U(VI) on Chinese soft-shelled turtle embryo fibroblasts (CSSTEF). Cell toxicity assay results showed that exposure to both Cr(VI) and U(VI) reduced the viability of CSSTEF cells, with half maximal inhibitory concentrations (IC₅₀) of 18.864 µM and 2.675 mM, respectively. Glutathione peroxidase activity was increased to 257.5 ± 94.6 U/mgprot under U(VI) exposure. Transmission electron microscopy showed mitochondrial damage in cells. Transcriptomic analysis indicated that both Cr(VI) and U(VI) exposure triggered the ribosome and oxidative phosphorylation pathways in CSSTEF cells, and affected glutathione metabolism, cardiac muscle contraction, and steroid biosynthesis pathways. In addition, U(VI) exposure affected the RNA polymerase and Notch signaling pathways. Collectively, these results demonstrated that both Cr(VI) and U(VI) exposure induced cytotoxicity, and affected protein synthesis and metabolic systems in CSSTEF cells. These findings provided new insights into the toxicological impacts of Cr(VI) and U(VI) exposure on Chinese soft-shelled turtle.</p>