The mitochondrial dysfunction in the pathogenesis of myocardial damage associated with high fat diet (HFD)-induced obesity remains largely unknown. Transmembrane protein 126B (TMEM126B), as a complex I assembly factor, plays a key role in regulating mitochondrial function. In the present study, the effects of TMEM126B on mitochondrial function were investigated using genetic knockout approach in HFD-induced mouse models with obesity. We found that TMEM126B was significantly increased in HFD-treated cardiac samples. Genetic ablation of TMEM126B alleviated HFD-mediated metabolic disorder and heart injury. TEM results suggested that cardiac mitochondrial integrity was improved in TMEM126B knockout mice compared with the wild type (WT) mice after HFD challenge. Additionally, the mitochondrial dysfunction induced by HFD was alleviated in mice with TMEM126B knockout, as evidenced by the decreased protein expression levels of dynamic-related protein-1 (DRP1) and fission-1 (FIS1) and increased expression of mitofusin-1 (MFN1). The mitochondrial impairments were further confirmed in palmitic acid (PA)-incubated cardiomyocytes, as evidenced by the down-regulated membrane potential and ATP levels, and by the up-regulated mitochondrial reactive oxygen species (ROS) production and DNA damage, which were significantly reversed by TMEM126B knockdown in vitro. Finally, TMEM126B ablation suppressed mitochondrial-dependent apoptotic death in the hearts of HFD mice. Therefore, TMEM126B led to mitochondrial impairments, contributing to the pathogenesis of HFD-induced cardiac injury, and blockage of TMEM126B could inhibit mitochondrial dysfunction, paving the road to new therapeutic modalities for the prevention of obesity-associated heart injury.