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Background Trichinellosis is a serious food-borne parasitic zoonosis worldwide. In the effort to develop vaccine against Trichinella infection, we have identified Trichinella spiralis Heat shock protein 70 (Ts-Hsp70) elicits partial protective immunity against T. spiralis infection via activating dendritic cells (DCs) in our previous study. This study aims to investigate whether DCs were activated by Ts-Hsp70 through TLR2 and/or TLR4 pathways. Methods and findings After blocking with anti-TLR2 and TLR4 antibodies, the binding of Ts-Hsp70 to DCs was significantly reduced. The reduced binding effects were also found in TLR2 and TLR4 knockout (TLR2-/- and TLR4-/-) DCs. The expression of TLR2 and TLR4 on DCs was upregulated after treatment with Ts-Hsp70 in vitro. These results suggest that Ts-Hsp70 is able to directly bind to TLR2 and TLR4 on the surface of mouse bone morrow-derived DCs. In addition, the expression of the co-stimulatory molecules (CD80, CD83) on Ts-Hsp70-induced DCs was reduced in TLR2-/- and TLR4-/- mice. More evidence showed that Ts-Hsp70 reduced its activation on TLR2/4 knockout DCs to subsequently activate the naïve T-cells. Furthermore, Ts-Hsp70 elicited protective immunity against T. spiralis infection was reduced in TLR2-/- and TLR4-/- mice correlating with the reduced humoral and cellular immune responses. Conclusion This study demonstrates that Ts-Hsp70 activates DCs through TLR2 and TLR4, and TLR2 and TLR4 play important roles in Ts-Hsp70-induced DCs activation and immune responses., Author summary Trichinellosis is a serious food-borne parasitic zoonosis caused by tissue-dwelling nematode Trichinella spiralis. Vaccine development is needed as an alternative approach to control the infection in domestic livestock or in humans. Ts-Hsp70 has been identified to elicit partial protective immunity against Trichinella spiralis infection via activating dendritic cells (DCs) in our previous study. This study aims to investigate the pathway(s) through which the Ts-Hsp70 activates DCs. Our results identified that Ts-Hsp70 could bind to DCs which was inhibited by blocking TLR2 and TLR4 with antibodies or TLR2 and TLR4 knockout. Ts-Hsp70 stimulated the expression of TLR2 and TLR4 and the co-stimulatory CD80, CD83 and CD86 on the surface of DCs which was reduced in TLR2 or TLR4 knockout mice. With TLR2 or TLR4 knockout, DCs were less stimulated by Ts-Hsp70 and subsequently reduce the activation of naïve T-cells. The protective immunity induced by Ts-Hsp70 against T. spiralis infection was also reduced in TLR2 or TLR4 knockout mice. The results conclude that Ts-Hsp70 activates DCs through activating TLR2 and TLR4 and TLR2 and TLR4 play important roles in Ts-Hsp70-induced protective immunity against Trichinella infection.