Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits, and Gα has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Gα, GDP is released, and GTP interacts to Gα. The GTP-bound activated Gα dissociates from GEF and Gβγ, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Gα subunits are subcategorized into four subfamilies: Gαi/o, Gαs, Gαq/11, and Gα12/13. Although the Gαi/o subtype family proteins, Gαi3 and GαoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GαoA possessing faster rates than Gαi3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The Gα subunit consists of a Ras-like domain (RD) and an α-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of Gαi3 and the AHD of GαoA showed a GDP/GTP turnover profile similar to that of GαoA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Gα subfamily.