Fatty acid binding proteins (FABPs) are members of the lipid binding protein superfamily and play crucial role in fatty acid transport and lipid metabolism. In macrophages, Adipocyte-type FABP is an important mediator of inflammation. However, the immune functions of FABPs in invertebrates are not well understood; here, we obtained the gene structure of Eriocheir sinensis FABP 3 and FABP 9 (EsFABP 3 and EsFABP 9), and compared with EsFABP 10. The mRNA expression profiles show that all three FABPs were significantly up-regulated in hemocytes after being challenged with bacteria. Of the three, EsFABP 3 was the most stable and also the most highly up-regulated. Further studies showed that knockdown of EsFABP 3 led to higher bacterial counts in the hemocyte culture medium and a significant decrease in the mRNA expression of some antimicrobial peptides following bacterial stimulation. Moreover, a subcellular study demonstrated that EsFABP 3 can affect nuclear translocation of the dorsal after Gram-positive bacterial stimulation in hemocytes. These findings support the notion that EsFABP 3 could inhibit bacterial proliferation by regulating antimicrobial peptides expression via the Toll signaling pathway.