Heme oxygenase-1 (HO-1) is an inducible and cytoprotective enzyme that provides a defense against oxidant damage. The present study screened 137 HO-1/interacting proteins using a profound co-immunoprecipitation (Co-IP) coupled with proteomics, and profiled the global HO-1 interactome network, including oxidative phosphorylation, endoplasmic reticulum and transport vesicle functions. Among these molecules, we observed that a novel interactor, emopamil-binding protein (EBP), is closely related to the cholesterol metabolism process. This study demonstrated that cholesterol promotes excessive oxidative stress and alters the energy metabolism in cardiomyocytes, further triggering numerous cardiovascular diseases. We observed that cholesterol caused the overexpression of EBP and HO-1 by the activation of AKT and Nrf2/mTOR pathways. In addition, HO-1 and EBP performed a myocardial protective function. The overexpression of HO-1 alleviated the cholesterol-induced excessive oxidative stress status by inhibition of the carbohydrate metabolism. Notably, we also confirmed that the loss of partial HO-1 activity aggravated the oxidative damage and cardiac systolic function induced by a high-fat diet in HO-1 heterozygous (HO-1+/-) mice. These findings indicate that the HO-1/EBP interaction plays a protective role in alleviating the dysfunction of oxidative stress and cardiac systolic function induced by cholesterol stimulation.