Abstract: Stimulator of interferon gene (STING) is an important adaptor protein in the innate immune signaling pathway, which mediates the production of type I IFNs. To study the role of STING in the innate immunity of Chinese soft-shelled turtle, Pelodiscus sinensis, the PsSTING gene cDNA was cloned. The result showed that the sequence was 2145 bp in full length, containing 1152 bp open reading frame and encoding 383 amino acids. The amino acid sequence alignment showed that, the N-terminal sequence of PsSTING protein contained 4 transmembrane regions and 2 endoplasmic reticulum retention motifs and the C-terminal sequence of PsSTING protein contained 1 helicase domain. The homology comparison and the phylogenetic tree showed that the PsSTING protein had a higher homology with the other species in the order Testudines, such as Platysternon megacephalum, followed by the homology with the Crocodilia and Avian, the lowest origin compared with invertebrates. The qRT-PCR results indicated that PsSTING was expressed in the all examined tissues including heart, liver, spleen, lung, kidney, small intestine, stomach, skin, muscle and blood cells in the Chinese soft-shelled turtle, and it was expressed at the highest level in the spleen tissue, followed by the liver, small intestine and lungs, with the lowest expression in blood cells. Compared with the control group, PsSTING was significantly up-regulated and then down-regulated after stimulation with Aeromonas hydrophila, LPS and poly(I:C). It was significantly higher than that in the control group at 12 hours post-infection (hpi), and reached the highest value at 24 hpi, and then gradually returned to the initial level after 48 hpi. Similarly, the protein expression of PsSTING was significantly increased in the spleen and the small intestine. The siRNA interference result demonstrated that the expression of PsSTING was significantly down-regulated in the small intestine (P＜0.05) and the expression levels of its downstream genes TBK1, IRF3, IRF7, STAT6 and IFN-β decreased significantly too after the interference of PsSTING. Its overexpression in HEK293T cells could significantly activate the promoter of pIFN-β-luc. The results of the study indicate that PsSTING participates in the regulation of the innate immunity of the Chinese soft-shelled turtle.