MicroRNA-33 (miR-33) and the SREBP host genes work together to control cholesterol homeostasis. SREBP proteins are key regulators of cholesterol biosynthesis and uptake. miR-33, located within the introns of SREBP genes, targets the ATP-binding cassette transporter A1 (ABCA1) for post-transcriptional repression. ABCA1 is crucial for high-density lipoprotein (HDL) synthesis and reverse cholesterol transport. Antisense inhibition of miR-33 increases ABCA1 expression and cholesterol efflux, while miR-33 overexpression decreases ABCA1 levels. In mice, injection of LNA-antisense oligonucleotides against miR-33 increases plasma HDL levels. These findings suggest that miR-33 acts in concert with SREBP host genes to regulate cholesterol homeostasis. miR-33 may be a therapeutic target for treating cardiometabolic diseases. SREBP and miR-33 are co-regulated by cholesterol, and miR-33 regulates ABCA1 expression, which is essential for cholesterol efflux and HDL synthesis. Manipulation of miR-33 levels affects cholesterol efflux from macrophages, and in vivo studies show that miR-33 antisense treatment increases HDL levels in mice. These results indicate that miR-33 and SREBP host genes work together to control cholesterol homeostasis, and miR-33 may be a potential therapeutic target for improving cholesterol metabolism in cardiometabolic diseases.MicroRNA-33 (miR-33) and the SREBP host genes work together to control cholesterol homeostasis. SREBP proteins are key regulators of cholesterol biosynthesis and uptake. miR-33, located within the introns of SREBP genes, targets the ATP-binding cassette transporter A1 (ABCA1) for post-transcriptional repression. ABCA1 is crucial for high-density lipoprotein (HDL) synthesis and reverse cholesterol transport. Antisense inhibition of miR-33 increases ABCA1 expression and cholesterol efflux, while miR-33 overexpression decreases ABCA1 levels. In mice, injection of LNA-antisense oligonucleotides against miR-33 increases plasma HDL levels. These findings suggest that miR-33 acts in concert with SREBP host genes to regulate cholesterol homeostasis. miR-33 may be a therapeutic target for treating cardiometabolic diseases. SREBP and miR-33 are co-regulated by cholesterol, and miR-33 regulates ABCA1 expression, which is essential for cholesterol efflux and HDL synthesis. Manipulation of miR-33 levels affects cholesterol efflux from macrophages, and in vivo studies show that miR-33 antisense treatment increases HDL levels in mice. These results indicate that miR-33 and SREBP host genes work together to control cholesterol homeostasis, and miR-33 may be a potential therapeutic target for improving cholesterol metabolism in cardiometabolic diseases.