The immunity-related GTPases (IRGs) are a family of proteins that play critical roles in innate resistance to intracellular pathogens. The number and diversity of IRG genes differ greatly in different species. Although IRG proteins have been well studies in mammals, they remain poorly characterized in lower vertebrates. In this study, we cloned two IRG genes, PsIRG5 and PsIRG8, from the Chinese soft-shelled turtle and compared their characterization and functional activity with mammalian IRGs. The PsIRG5 is a gene of 1896 bp that encodes a protein of 413 amino acid and PsIRG8 is 1543 bp in length encoding another 413 aa protein. Sequence alignment between all turtle IRG-like genes and mammalian IRGs showed that both PsIRG5 and PsIRG8 were conserved with mammalian GKS IRGs, while PsIRG5 appeared a closer evolutionary relationship with mammalian GMS IRGs. The expression and subcellular characterization revealed that PsIRG5 was dramatically upregulated under Aeromonas hydrophila challenge and exhibited co-localization with lysosomes in cells; whereas PsIRG8 was downregulated and has no distinct localization. Functional activity assay demonstrated that PsIRG5 plays a role in autophagy induction and IFN-γ contributes to enhance the induction, since it has IFN-inducible elements in its promoter region. These data above unravel the molecular characterization and functional activity of IRGs in lower vertebrate for the first time and will provide insights into the comparative immunity and evolutionary relationships of IRGs between mammals and reptiles.