The role of nitric oxide synthase 1 (NOS1) as a major modulator of cardiac function has been extensively studied in experimental models; however, its role in human ischemic cardiomyopathy (ICM) has never been analysed. Thus, the objectives of this work are to study NOS1 and NOS-related counterparts involved in regulating physiological function of myocyte, to analyze NOS1 localisation, activity, dimerisation, and its relationship with systolic function in ICM. The study has been carried out on left ventricular tissue obtained from explanted human hearts. Here we demonstrate that the upregulation of cardiac NOS1 is not accompanied by an increase in NOS activity, due in part to the alterations found in molecules involved in the regulation of its activity. We observed partial translocation of NOS1 to the sarcolemma in ischemic hearts, and a direct relationship between its protein levels and systolic ventricular function. Our findings indicate that NOS1 may be significant in the pathophysiology of human ischemic heart disease with a preservative role in maintaining myocardial homeostasis.