C-type lectins (CTLs) have received widespread attention in animal immune responses. In the present study, two CTLs ( and ) were identified from obscure puffer . The open reading frames of ToCTL1 and ToCTL2 were 687 and 1,380 bp, respectively. The predicted ToCTL1 and ToCTL2 proteins contained a single transmembrane region and one typical carbohydrate recognition domain (CRD). Quantitative real-time polymerase chain reaction detected and transcripts in all examined tissues, with high levels in the intestine and kidney, and their expression levels were remarkably altered upon and infection. The recombinant proteins ToCTL1-CRD and ToCTL2-CRD agglutinated the Gram-negative and Gram-positive bacteria in a Ca-dependent manner. rToCTL1-CRD and rToCTL2-CRD exhibited evident binding activities against seven kinds of bacteria and polysaccharides (lipopolysaccharide and peptidoglycan) in a Ca-independent manner. Moreover, rToCTL1-CRD and rToCTL2-CRD could inhibit the growth of four types of bacteria . These findings collectively demonstrated that ToCTL1 and ToCTL2 could be involved in host defense against bacterial infection in .