Toll-interacting protein (Tollip) is a critical regulator of Toll-like receptor (TLR)-mediated innate immune responses. However, the Tollip gene has not been systematically characterized in shellfish. In this study, we identified and characterized a Tollip gene, PyTollip, in Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine its sequence identities and evolutionary relationships. Compared with Tollip genes from other invertebrate and vertebrate species, the PyTollip gene is highly conserved in its sequence and structural features, except that a unique asparagine residue was found at a conserved site in the C2 domain of PyTollip. Quantitative real-time PCR was used to investigate the expression profiles of PyTollip in different developmental stages, healthy adult tissues, and in hemolymph after Micrococcus luteus and Vibrio anguillarum bacterial infection. Real-time PCR analysis demonstrated differential expression of PyTollip at the acute phase (3 h) after infection with Gram-negative (V. anguillarum) and Gram-positive (M. luteus) bacteria. A second strong response of PyTollip expression was observed 24 h after challenge with V. anguillarum. Collectively, these results provide novel insights into the specific role and response of Tollip and TLR signaling pathways in host immune responses against different bacterial pathogens in bivalves.