As an important economic shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahaemolyticus. Similar to other invertebrates, Haliotis discus hannai resists pathogen infection by innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinases (MMPs) family. As MMP-1 was engaged in the innate immunity of abalone, the involvement of TIMP in the regulation of MMP-1 expression and activity as well as the immune responses merits further investigation. In this study, the role of TIMP in the innate immune responses of H. discus hannai against V. parahaemolyticus infection and the interaction between TIMP and MMP-1 were investigated. The full length cDNA sequence of TIMP was obtained, and the expressions of TIMP in different tissues of H. discus hannai were analyzed post Vibrio infection. The cloned TIMP cDNA sequence was 2291 bp in length. NetNGlyc 1.0 Server and Netoglyc 4.0 Server were used to analyze the glycosylation sites of TIMP. The results showed that TIMP had three potential N-glycosylation sites, namely Asn at positions 47, 77 and 152, and a potential O-glycosylation site, namely Thr at position 108. Multiple alignment of TIMP amino acid sequences from different molluscs showed that the similarities of TIMP in H. discus hannai with those of H. diversicolor, Crassostrea gigas and Tegillarca granosa were 76%, 18.9% and 19.3%, respectively. In the early stage of V. parahaemolyticus infection, the expression of TIMP in hemocytes and gill tissues were significantly up-regulated. To study the interaction between TIMP and MMP-1 in the anti-Vibrio immunity of abalone, RNA interference technology was used to knock down the expression levels of TIMP and MMP-1. After the silencing of MMP-1 gene in abalone, TIMP expression was significantly up-regulated. When TIMP expression was inhibited, MMP-1 expression decreased significantly. These results indicated that MMP-1 expression was positively regulated by TIMP, while TIMP expression was negatively regulated by MMP-1 in abalone. In conclusion, the present study will help to reveal the interaction between TIMP and MMP-1 in abalone innate immunity against V. parahaemolyticus infection. It also provided new insights into the prevention and control of pathogens in abalone culture.