Mitochondrial sterol 27-hydroxylase (CYP27A) plays a major role in macrophage cholesterol homeostasis by metabolizing cholesterol to polar oxysterols, which can ligate liver X receptor α (LXRα) and upregulate genes involved in cholesterol efflux including ATP binding cassette transporter A1 (ABCA1). Here, we exploit the cholesterol trafficking properties of Steroidogenic Acute Regulatory protein (StAR; StarD1), to increase the rate-limiting step of cholesterol transport to the inner mitochondrial membrane where CYP27A resides, and describe the impact of this on macrophage cholesterol efflux mechanisms. Stably transfected RAW 264.7 macrophages, containing either pCMV.5 or pCMV.5_StarD1, were generated by hygromycin selection. Murine StarD1 amplicon (355bp) was detected by PCR only in cells transfected with pCMV.5_StarD1; sequencing of this fragment confirmed its identity. Mature StarD1 protein (30kDa) was detected only in lysates from macrophages transfected with pCMV.5_StarD1, and not in empty vector controls, or wild type RAW 264.7 macrophages. Expression of StAR significantly enhanced (1.5 fold; p<0.001) macrophage cholesterol efflux to apoAI (20μg ml-1), but not HDL, in the presence of dibutyryl cAMP [(Bu)2cAMP (0.3mM)], compared to empty vector, or wild type controls. Macrophages stably transfected with mutated murine StAR (Arg181Leu), which abrogates the mitochondrial cholesterol trafficking properties of this protein, did not show any enhancement of cholesterol efflux. Inhibition of CYP27A1, using GW2739297x (1μM), blocked the impact of StAR on apoAI-dependent cholesterol efflux. Further, treatment with the LXR antagonist (geranyl geranyl pyrophosphate, ammonium salt) negated the effect of StAR. At the end of the efflux period (24h), cellular levels of ABCA1 mRNA and protein were lower in cells expressing StAR, compared with cells expressing the empty vector, consistent with lower cholesterol content and higher cholesterol efflux from these cells. No changes in expression of ABCG1, ABCG4 or ABCA7 relative to GAPDH were detected by PCR in either cell type. Thus, introduction of StAR into macrophages exerts a markedly beneficial influence upon cholesterol efflux mechanisms, and may provide an avenue for the development of anti-atherogenic therapeutics.