Lipopolysaccharide (LPS) is one of the many reasons that can cause myocardial injury. Our previous works have demonstrated that 14-3-3γ could protect myocardium against LPS-induced injury. Tetramethylpyrazine (TMP), an alkaloid found in Chinese herbs, exerts myocardial protection in many ways with multiple targets. We hypothesized that the cardioprotection of TMP against LPS-induced injury is attributed to upregulation of 14-3-3γ and improvement of mitochondrial function. To test the hypothesis, we investigated the effects of TMP on LPS-induced injury to cardiomyocytes by determining cell viability, LDH and caspase-3 activities, reactive oxygen species and MMP levels, mPTP openness, and apoptosis rate. The expression of 14-3-3γ and Bcl-2, and the phosphorylation of Bad (S112) were examined by Western blot. LPS-induced injury to cardiomyocytes was attenuated by TMP via upregulating expression of 14-3-3γ, and Bcl-2 on mitochondria, activating Bad (S112) phosphorylation, increasing cell viability and MMP levels, decreasing LDH and caspase-3 activity, reactive oxygen species generation, mPTP opening and apoptosis rate. However, the cardioprotection of TMP was attenuated by pAD/14-3-3γ-shRNA, an adenovirus that knocked down intracellular 14-3-3γ expression. In conclusion, the cardioprotection of TMP against LPS-induced injury was through up-regulating the expression of 14-3-3γ, promoting the translocation of Bcl-2 to mitochondria, and improving the function of mitochondria.