Nitric oxide (NO) is a key effector molecule produced in the innate immune systems of many species for antimicrobial defense. However, how NO production is regulated during bacterial infection in invertebrates, especially crustaceans, remains poorly understood. Vibrio harveyi, a Gram-negative marine pathogen, is among the most prevalent and serious threats to the world's shrimp culture industry. Its virulence typically manifests itself through shrimp hepatopancreas destruction. In the current study, we found that NO generated by an in vitro donor system (NOC-18) could rapidly and effectively kill V. harveyi. In addition, injection of heat-killed V. harveyi increased the concentration of NO/nitrite and the mRNA expression of nitric oxide synthase (NOS) in the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), the commercially most significant shrimp species. Live V. harveyi challenge also induced NO/nitrite production and NOS gene expression in primary L. vannamei hepatopancreatic cells in a time- and dose-dependent manner. Co-incubation of l-NAME, an inhibitor selective for mammalian constitutive NOSs, dose-dependently blocked V. harveyi-induced NO/nitrite production, without affecting V. harveyi-induced NOS mRNA expression. Furthermore, l-NAME treatment significantly increased the survival rate of infecting V. harveyi in cultured primary hepatopancreatic cells of L. vannamei. As a whole, we have demonstrated that endogenous NO produced by L. vannamei hepatopancreatic cells occurs in enzymatically regulated manners and is sufficient to act as a bactericidal molecule for V. harveyi clearance.