Planarian, a representative of platyhelminthes, has strong regeneration ability and less complicated innate immune system. However, planarian immune system remains poorly understood. In this paper, a novel C-type lectin-like protein, namely, DjCTL was identified and characterized in Dugesia japonica. DjCTL was mainly expressed in the pharyngeal and epidermis and up-regulated upon the induction of lipopolysaccharide (LPS), peptidoglycan (PGN), Gram-positive and Gram-negative bacteria indicating that DjCTL may be involved in the immune responses. Recombination DjCTL protein agglomerated rabbit red blood cells and interacted with LPS, PGN, mannose and galactose as well as both Gram-positive and Gram-negative bacteria, but it can only cause the agglutination of Gram-negative bacteria. Importantly, in the early periods of regeneration, DjCTL had a significantly high expression and was mainly expressed in early blastemas. RNA interference of DjCTL by dsRNA-DjCTL led to a slow wound healing during regeneration. These findings suggest that DjCTL participates in the innate immune response and plays an important role in early stages of regeneration.