The study investigates the role of miR-33, an intronic microRNA located within the gene encoding sterol-regulatory element–binding factor–2 (SREBF-2), in regulating cholesterol homeostasis. MiR-33 is shown to modulate the expression of genes involved in cellular cholesterol transport, particularly by inhibiting the adenosine triphosphate–binding cassette (ABC) transporter ABCA1 and the cholesterol transporter ABCG1. In mouse and human cells, miR-33 reduces ABCA1 expression, leading to decreased cholesterol efflux to apolipoprotein A1 and high-density lipoprotein (HDL). Lentiviral delivery of miR-33 in mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 increases hepatic ABCA1 expression and plasma HDL levels. The study also identifies ABCA1 and NPC1 as conserved targets of miR-33, while ABCG1 is a target only in mice. These findings suggest that miR-33 plays a crucial role in regulating both HDL biogenesis in the liver and cellular cholesterol efflux, providing a potential therapeutic target for enhancing HDL levels and reducing atherosclerotic risk.The study investigates the role of miR-33, an intronic microRNA located within the gene encoding sterol-regulatory element–binding factor–2 (SREBF-2), in regulating cholesterol homeostasis. MiR-33 is shown to modulate the expression of genes involved in cellular cholesterol transport, particularly by inhibiting the adenosine triphosphate–binding cassette (ABC) transporter ABCA1 and the cholesterol transporter ABCG1. In mouse and human cells, miR-33 reduces ABCA1 expression, leading to decreased cholesterol efflux to apolipoprotein A1 and high-density lipoprotein (HDL). Lentiviral delivery of miR-33 in mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 increases hepatic ABCA1 expression and plasma HDL levels. The study also identifies ABCA1 and NPC1 as conserved targets of miR-33, while ABCG1 is a target only in mice. These findings suggest that miR-33 plays a crucial role in regulating both HDL biogenesis in the liver and cellular cholesterol efflux, providing a potential therapeutic target for enhancing HDL levels and reducing atherosclerotic risk.