Cholesterol homeostasis results from a delicate interplay between influx and efflux of free cholesterol primarily mediated by ABCA1. Here we report downregulation of ABCA1 in hyper-cholesterol conditions in macrophages, which might be responsible for compromised reverse cholesterol transport and hyperlipidemia. Surprisingly, this is countered by the upregulation of a lesser known family member ABCA5 to maintain cholesterol efflux. The relative contribution of ABCA1 and ABCA5 toward cholesterol efflux was evaluated and revealed ABCA5 as the primary efflux mediator under high cholesterol load. These observations were correlated to cholesterol load in circulation in vivo , and we observed an inverse expression profile in mice models of atherosclerosis (ApoE−/−) and hyperlipidemia (PPARα−/−) in response to high cholesterol diet. Observations were further validated in human plasma samples. Simulation studies revealed a unique conformation of ABCA5 proposing a favored route for cholesterol loading onto high-density lipoproteins for reverse cholesterol transport. Thus, our study implicates a functional complementation between these two transporters, formulating an efficient strategy to maintain efflux in cholesterol excess conditions in macrophages. Unlabelled Image • The efflux of excess cholesterol in macrophages, primarily mediated by ABCA1, is essential for alleviating atherosclerosis, but the role of secondary transporters has not been explored in details. • The report demonstrates the efflux coordination by different mechanisms depending on the cholesterol concentration and the emergence of ABCA5 as the primary efflux mediator to counter the reduction in ABCA1 function under hyper-cholesterol conditions. • Plasma circulatory levels of ABCA1 and ABCA5 present a novel indicator of understanding cholesterol levels and their clinical correlation to reverse cholesterol transport. • Our study establishes ABCA5 as a cholesterol efflux mediator with special functional role in high cholesterol conditions through a conformational change which offsets the reduction in ABCA1 expression. [ABSTRACT FROM AUTHOR]