Edwardsiella piscicida is a pathogenic bacterium, which can infect a number of fish species and cause a disease termed edwardsiellosis, threatening global fish farming with high prevalence and mortality. Thiamine (Vitamin B1), functioning in the form of thiamine pyrophosphate (TPP), is essential for almost all organisms. Bacteria acquire TPP by biosynthesis or by transportation of exogenous thiamine. TPP availability has been associated with bacterial pathogenicity, but the underlying mechanisms remain to be discovered. The role of thiamine in the pathogenicity of E. piscicida is unknown. In this study, we characterized a thiamine transporter (TT) operon in E. piscicida. The deletion of the TT operon resulted in an intracellular TPP lacking situation, which led to attenuated overall pathogenicity, impaired abilities associated with motility and host cell adhesion, as well as decreased expression of certain flagellar and adhesion genes. Moreover, TPP starvation led to intracellular c-di-GMP reduction, and introducing into the TPP-suppressed mutant strain an exogenous diguanylate cyclase for c-di-GMP synthesis restored the virulence loss. Taken together, this work reveals the involvement of thiamine uptake in the virulence regulation of E. piscicida, with c-di-GMP implicated in the process. These finding could be employed to explore potential drug targets against E. piscicida.