Urease, a nickel-dependent enzyme, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Maturation of urease involves the proper insertion of nickel to its active site and requires at least 4 accessory proteins, namely, UreE, UreF, UreG, and UreD. In addition, it was also demonstrated that both metallochaperones UreE and UreG play a critical role in the maturation of apo-urease. Previously, we obtained the predominant urease gene cluster from ruminal uncultured bacteria BIN257 by metagenomic sequencing and binning. In this study, we overexpressed and purified UreG and UreE from BIN257, nickel binding to UreG and its interactions with UreE as well as their roles in Ni²⁺ transfer were studied. We demonstrated by pull-down assays that UreE could combine with UreG in vitro. The properties of UreE-UreG interactions were examined using isothermal titration calorimetry (ITC) and analytical ultracentrifuge (AUC). The ITC data revealed that the reaction of UreE and UreG was an endothermic process dominated by hydrophobic action. Moreover, the stoichiometry of this band was 1:1, and AUC experiments showed that molecule masses of UreE and UreG were ~51.5 and 25.2 kDa, respectively, corresponding to UreE dimer and an UreG monomer, indicating that each dimer of UreE binds 2 molar eq of UreG monomer to format a complex (UreE)₂-(UreG)₂. That conformational changes are essential for UreG to change protein partners were further detected by mutagenesis and AUC studies. We identified that UreG residues Glu-23, Asp-41, Glu-46, Glu-66, Cys-70, His-72, Asp-78, and Asp-118 played crucial roles in the conformational changes upon nickel and UreE binding. ITC experiments revealed that Glu-23, Asp-41, Glu-46, Glu-66, Cys-70, His-72, and Asp-78 on UreG were critical for nickel transfer from UreE to UreG, and Glu-66, Cys-70, His-72, and Asp-78 were also necessary to combine with UreE for UreG. Based on our results, interactions and nickel transfer between UreE and UreG play an important role in urease maturation process.