Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO22+) to interact with various proteins and disturb their biological functions. Cytochrome b 5 (cyt b 5 ) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b 5 -cyt c complex. In previous molecular modeling study in combination with UV–Vis studies, we found that UO22+ is capable of binding to cyt b 5 at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b 5 and cyt c, as well as cyt b 5 -cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b 5 (K D =10μM), cyt c (K D =87μM), and cyt b 5 -cyt c complex (K D =30μM) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b 5 -cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b 5 /cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity.