Cardiac contractility is controlled by key sarcoplasmic reticulum (SR) Ca2+ handling proteins that regulate intracellular Ca2+ homeostasis. Impaired Ca2+ handling has been associated with heart failure, thus emphasising its critical role in cardiac function. During cardiac relaxation, SR Ca2+ uptake is regulated by the activity of the sarco(endo)plasmic reticulum Ca2+ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN). We have identified HS-1 associated protein X-1 (HAX-1), a ubiquitously expressed mitochondrial protein with anti-apoptotic function, as a novel binding partner of PLN. This association was found to cause a subcellular redistribution of HAX-1 from mitochondria to the ER and enhanced the protective effects of HAX-1 on cell survival. Deletion analysis determined that a C-terminal fragment encompassing amino acids 182–279 of HAX-1 were required for binding to PLN and ER localisation, while mitochondrial targeting required an N- terminal region of the protein. HAX-1 overexpression in HEK293 cells resulted in significant reduction of ER Ca2+ content, providing evidence for a role in promoting cell survival through modulation of ER Ca2+ stores. Moreover, HAX-1 overexpression in cardiomyocytes was associated with decreased SR Ca2+ content and depressed cardiomyocyte contractility. Our recent findings suggest a protective effect of HAX-1 on mitochondria as indicated by increased resistance to swelling induced by calcium and depressed cytochrome c release following oxidative stress in mitochondria isolated from HAX-1 overexpressing hearts. Collectively, our findings provide insights on the mechanisms through which HAX-1 may modulate cell survival, indicating its importance in cardiac muscle.