Objective：To screen for an optimized siRNA sequence targeting rat Toll-like receptor 4 (TLR4) in vitro. Methods: The full length gene of rat TLR4 was cloned and inserted into pEGFP-C1 plasmid to construct pEGFP-rTLR4. Three pairs of siRNAs targeting rTLR4 were chemically synthesized and were co-transfected with pEGFP-rTLR4 into HEK-293 cells via Lipofectamine2000. Cells were also co-transfected with siRNA targeting EGFP and negative control siRNA. The expression of EGFP was observed under inverted fluorescene microscope and flow cytometry. Results: Compared with the negative control group, 3 pairs of siRNAs targeting TLR4 and one pair of siRNA targeting EGFP significantly suppressed the EGFP expression (P<0.05); the inhibitory effect of siRNA2（gene sequence:5′-GTC TCA GAT ATC TAG ATC T-3′,19 bp, 1 352-1 370）was the strongest one, with an interference efficiency over 75%.Conclusion: We have successfully obtained the siRNA sequence targeting TLR4 mRNA, which can efficiently suppress the expression of rat TLR4 mRNA in vitro.