Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain, although a Toll-like receptor (SpToll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 (SpToll2) from S. paramamosain. The full-length cDNA of SpToll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that SpToll2 clustered with Drosophila Toll1, and shared high homology with PtToll4. Real-time qPCR analysis showed that SpToll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both SpToll1 and SpToll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus, Staphylococcus aureus, Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant SpToll1-LRR and SpToll2-LRR proteins could bind to V. parahaemolyticus, S. aureus, Escherichia coli, and Beta Streptococcus. In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of SpAMPs after the challenges with V. parahaemolyticus or S. aureus. The data suggested that SpToll1and SpToll2 could regulate the transcripts of several AMPs and four immune related mediators (SpMyD88, SpTube, SpPelle and SpTRAF6) at different scale. While silencing of SpToll1 post pathogens challenge attenuated the expression of SpHistin, SpALF1 and SpALF5 in mud crab's hemocytes, depletion of SpToll2 post pathogens challenge inhibited the expression of SpALF1-6, SpGRP, SpArasin and SpHyastastin. Furthermore, the results of overexpression assay also showed SpToll1 and SpToll2 could enhance the promoter activities of SpALFs in mud crab. Taken together, these results indicated that SpToll1 and SpToll2 might play important roles in host defense against pathogen invasions in S. paramamosain.