Macrophage-specific overexpression of cholesteryl ester hydrolysis in hormone-sensitive lipase transgenic (HSL Tg) female mice paradoxically increases cholesterol esterification and cholesteryl ester accumulation in macrophages, and thus susceptibility to diet-induced atherosclerosis compared to nontransgenic C57BL/6 mice. The current studies suggest that whereas increased cholesterol uptake could contribute to transgenic foam cell formation, there are no differences in cholesterol synthesis and the expression of cholesterol efflux mediators (ABCA1, ABCG1, apoE, PPARγ, and LXRα) compared to wild-type macrophages. HSL Tg macrophages exhibit twofold greater efflux of cholesterol to apoA–I in vitro, suggesting the potential rate-limiting role of cholesteryl ester hydrolysis in efflux. However, macrophage cholesteryl ester levels appear to depend on the relative efficacy of alternate pathways for free cholesterol in either efflux or re-esterification. Thus, increased atherosclerosis in HSL Tg mice appears to be due to the coupling of the efficient re-esterification of excess free cholesterol to its limited removal mediated by the cholesterol acceptors in these mice. The overexpression of cholesterol acceptors in HSL-apoA-IV double-transgenic mice increases plasma HDL levels and decreases diet-induced atherosclerosis compared to HSL Tg mice, with aortic lesions reduced to sizes in nontransgenic littermates. The results in vivo are consistent with the effective efflux from HSL Tg macrophages supplemented with HDL and apoA-I in vitro, and highlight the importance of cholesterol acceptors in inhibiting atherosclerosis caused by imbalances in the cholesteryl ester cycle.