Tripartite motif 8 (TRIM8) is a member of the TRIM protein family that has been found to be implicated in cardiovascular disease. However, the role of TRIM8 in myocardial ischemia/reperfusion (I/R) has not been investigated. We aimed to explore the effect of TRIM8 on cardiomyocyte H9c2 cells exposed to hypoxia/reoxygenation (H/R). We found that TRIM8 expression was markedly upregulated in H9c2 cells after stimulation with H/R. Gain- and loss-of-function assays proved that TRIM8 knockdown improved cell viability of H/R-stimulated H9c2 cells. In addition, TRIM8 knockdown suppressed reactive oxygen species production and elevated the levels of superoxide dismutase and glutathione peroxidase. Knockdown of TRIM8 suppressed the caspase-3 activity, as well as caused significant increase in bcl-2 expression and decrease in bax expression. Furthermore, TRIM8 overexpression exhibited apposite effects with knockdown of TRIM8. Finally, knockdown of TRIM8 enhanced the activation of PI3K/Akt signaling pathway in H/R-stimulated H9c2 cells. Inhibition of PI3K/Akt by LY294002 reversed the effects of TRIM8 knockdown on cell viability, oxidative stress, and apoptosis of H9c2 cells. These present findings defined TRIM8 as a therapeutic target for attenuating and preventing myocardial I/R injury.