Acute myocardial infarction (AMI) is the main cause of death in the whole world. This study aimed to investigate whether forkhead box O4 (FoxO4) could negatively modulate ubiquitin specific peptidase 10 (USP10) transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation (H/R)-induced cardiomyocytes through Hippo/YAP pathway. mRNA expression as well as protein expressions of USP10 and FoxO4 in H9C2 cells after H/R induction or transfection were respectively detected by Reverse transcription-quantitative (RT-q) PCR analysis and Western blot. The viability and apoptosis of H9C2 cells after H/R induction or transfection were respectively detected by CCK-8 and TUNEL assays. The expressions of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in H9C2 cells after H/R induction or transfection were analyzed using appropriate kits and intracellular reactive oxygen species (ROS) levels were detected using a ROS Assay Kit. Dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP) have adopted to confirm the combination of USP10 and FoxO4. Western blot was also used to analyze the expression of apoptosis-related proteins and Hippo/YAP pathway-related proteins. As a result, USP10 expression was decreased in H/R-induced H9C2 cells in a time-dependent manner. USP10 overexpression increased the viability and suppressed the apoptosis and oxidative stress of H/R-induced H9C2 cells. In addition, FoxO4 modulated USP10 transcription. FoxO4 expression was increased in H9C2 cells induced by H/R. FoxO4 overexpression could reverse the protective effects of USP10 overexpression on H/R-induced H9C2 cells by regulating the Hippo/YAP signaling pathway. In conclusion, FoxO4 negatively modulated USP10 transcription to aggravate the apoptosis and oxidative stress of H/R-induced H9C2 cells via blocking Hippo/YAP pathway.