Introduction The first line of invertebrate defense mechanism is believed to rely on innate immunity to limit infection by pathogens. The innate immune system consists of physical, humoral and especially cellular responses, which play a crucial role in phagocytosis, encapsulation and nodule formation. In addition, the humoral barrier can also synthesize many antimicrobial peptides, pathogen recognition receptors (PRRs), prophenol oxidase, and coagulation cascade to eradicate pathogens [1–3]. However, all mechanisms must act in a concerted manner to ensure an effective response. Consequently, signaling pathways are an important mechanism for regulating the cooperation of immune responses. Calcium ion (Ca2+), one of the most essential intracellular second messengers, plays a crucial role in the signaling pathway to activate many biological functions including the immune response. Signal transduction is induced by the alteration of intracellular Ca2+ concentration which depends on various stresses such as pathogen infection [4-5]. Calmodulin (CaM), a ubiquitous intracellular Ca 2+ sensor, is a small (∼17 kDa) signaling protein that contains N- and C-terminal lobes that connect with a flexible central linker. Each lobe is composed of two EF hand motifs, helix-loop-helix structure, with two Ca2+ binding sites [6-7]. Ca2+ binding causes a conformational change of CaM by bringing two helices of each lobe into a more perpendicular conformation and exposing hydrophobic residues on their surface, which makes it able to interact with specific target proteins and regulate their functions [8] In marine invertebrates, CaM not only regulates Ca2+ metabolism in intracellular processes, but is thought to play a crucial role in extracellular processes, including biomineralization leading to shell formation in mussels [5]. Furthermore, it can regulate shrimp moulting in the crayfish, Procambarus clarkii [9]. In shrimp, CaM was first isolated from Crangon crangon in 1992. Their biological and physiological properties are similar to those of other invertebrate CaMs [10]. Furthermore, an alteration in the expression level of CaM gene and protein was detected in hemocytes of shrimp infected with pathogens in Litopenaeus vannamei and Penaeus monodon, respectively [4, 11–12]. It could be indicated that CaM might also relate to the defense mechanism of pathogens through signaling pathway. Although CaM-mediated processes have been extensively studied, the signal transduction involved in the defense mechanism of pathogens is still limited. In this study, the recombinant CaM protein from the black tiger shrimp, Penaeus monodon (PmCaM), was characterized to verify a secondary structure and monitor a conformational change upon calcium binding and identify CaM-binding proteins involved in hemocytes of shrimp. Additionally, the PmCaM gene was silenced to determine whether it affects the mortality of shrimp infected with Vibrio harveyi.