Reactive oxygen species (ROS) produced by NADPH oxidase 5 (Nox5) are regulated by Ca2+ flux through the interactions of its self-contained EF-hand domain (EFD), dehydrogenase domain (DH), and transmembrane domain. Studies suggest that the regulatory EF-hand binding domain (REFBD) and phosphorylatable (PhosR) sequences within DH play an important role in Nox5's superoxide-generating activity. However, the interplay of the EFD-DH interaction is largely unclear. Here, we used two synthetic peptides corresponding to the putative REFBD and PhosR sequences, as well as DH construct proteins, and separately studied their binding to EFD by fluorescence spectroscopy and calorimetry. With mutagenesis, we revealed that the C-terminal half domain of EFD binds specifically to REFBD in a Ca2+-dependent manner, which is driven primarily by hydrophobic interactions to form a more compact structure. On the other hand, the interaction between EFD and PhosR is not Ca2+-dependent and is primarily dominated by electrostatic interactions. The binding constants (Ka) for both peptides to EFD were calculated to be in the range of 105 M−1. The formation of the binary complex EFD/REFBD and ternary complex EFD/REFBD/PhosR was demonstrated by fluorescence resonance energy transfer (FRET). However, EFD binding to PhosR appears to be not biologically important while the conformational change on its C-terminal half domain resembles a major factor in EFD-DH domain-domain interactions.