Recent studies have initiated a paradigm shift in understanding heat shock protein 70 (HSP70) functions in the shrimp immune system. However, the mechanism by which Litopenaeus vannamei (Lv)HSP70 modulates the innate immune response remains unclear. This study shows that LvHSP70 binds to the pattern recognition receptor LPS and β-1,3-glucan–binding protein (LvLGBP), and subsequently leads to the activation of the prophenoloxidase system. Injection of shrimp with rLvHSP70 significantly (p < 0.05) upregulated the gene and protein expression of the key pattern recognition receptor LvLGBP. A coimmunoprecipitation and ELISA-based binding assay strongly confirmed the binding of LvHSP70 to LvLGBP at polysaccharide recognition motifs (PLS motifs) with a Kd of 4.44 μM and its competitive binding with LPS (IC50) is 8.036 μM. Conversely, LPS efficiently competed with LvHSP70 for binding to LvLGBP in a concentration-dependent manner with an IC50 of 7.662 μM, indicating that both are ligands of LvLGBP and likely bind at the same site. Binding of LvHSP70 to LvLGBP highly activated phenoloxidase activity in shrimp hemocyte lysate supernatants. Gene silencing of LvLGBP impaired the activation of phenoloxidase activity in shrimp by rLvHSP70, indicating that LvHSP70–LvLGBP interaction was essential for stimulating the immune cascade. Taken together, these results demonstrated that LvHSP70 is a ligand of LvLGBP similar to LPS and acts as a damage-associated molecular pattern to modulate the shrimp immune system via the prophenoloxidase system, eventually leading to the production of melanin and toxic reactive intermediates against invading pathogens.