Objective：To investigate the time correlation with the activation of molecule targets in Toll Like Receptor 4（TLR4） signaling in mice myocardium activated by lipopolysaccharide（LPS） and its significance. Methods：Male C57BL／6J mice were randomly divided into LPS group（intraperitoneal injection with LPS 10 mg／kg） and saline control group. The expression of molecule targets in TLR4 signaling parthway were detected at 5 min，10 min，30 min，1 h，2 h，6 h，12 h，24 h（n = 3） after treatment by Ｗestern blotting. Resultｓ：The expression of TLR4-MyD88-IκBα signaling is activated 5 min after injection, reached the peak at 2 h, then decreased（P < 0.05）. The expression of MAPKs is activated 5 min after injection, reached the peak at 6 h, then decreased（P < 0.05）. The expression of Akt is activated 10 min after injection, reached the peak at 6 h, then decreased（P < 0.05）. Conclusion： There is a close correlation between the activation of TLR4 signaling and the time of LPS treatment in mice myocardium .