Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.