RNA interference assays of three types of MrTLRs in Macrobrachium rosenbergii were conducted using specific short-interfering RNAs (siRNAs) with doses of 0.4, 0.8, and 1.2 μg/g, and the relative expression levels of three Toll-like receptor genes in gills were analyzed with relative quantitative real-time PCR. The result showed that 1.2 μg/g was the optimal dose of siRNA for RNA interference assay of MrTLRs in the present study. In the RNA interference assay of MrTLR1, the expression of Myeloid differentiation factor 88 (MyD88), Crustin and anti-lipopolysaccharide factor (ALF) in MrTLR1-silenced prawns obviously declined at 24 h and 48 h post injection, whereas the MrTLR2 expression was significantly upregulated. Furthermore, the expression levels of MyD88 and Crustin significantly decreased in MrTLR2-silenced prawns, and the silencing of MrTLR3 induced remarkable upregulation of the expression of immune deficiency (IMD) and ALF in M. rosenbergii. Additionally, there was no significant difference of prophenoloxidase (proPO) expression between MrTLRs-silenced groups and scrambled-siRNA group. These results suggested that MrTLR1 and MrTLR2 might be involved in the regulation of antibacterial peptides expression, and MrTLR3 exhibited regulatory effects on the Toll signaling pathway as well as IMD signaling pathway in M. rosenbergii.