Alpha-1-antitrypsin (AAT), a circulating serine protease inhibitor, is an acute inflammatory response protein with anti-inflammatory functions. The C-terminal peptides of AAT are found in various tissues and have been proposed as putative bioactive peptides with multiple functions, but its mechanism of action remains unclear. We previously reported that a mouse AAT C-terminal peptide of 35 amino acids (mAAT-C1-35) activates guanine nucleotide-binding protein subunit alpha 13 (Gα13). Here, we show that mAAT-C1-35 binds directly to the guanosine diphosphate (GDP)-bound form of Gα13 through the N-terminal region (mAAT-C1-17), thereby facilitating the association of Gα13・GDP with its downstream target proteins in the absence of GTP.