Niacin (nicotinic acid) is the most potent clinically used agent for increasing plasma HDL and apolipoprotein (apo) A-I. The mechanism by which niacin increases apoA-I is not clearly understood. We have examined the effect of niacin on the hepatic production and removal of apoA-I using Hep G2 cells as an in vitro model. Incubation of Hep G2 cells with niacin resulted in increased accumulation of apoA-I in the medium in a dose-dependent manner. Incorporation of [³H]leucine and [³⁵S]methionine into apoA-I and apoA-I mRNA expression was unchanged by niacin, suggesting that it did not affect apoA-I de novo synthesis. Uptake of radiolabeled HDL protein and HDL apoA-I by Hep G2 cells was significantly reduced to as much as 82.9±2.2% (P=.04) and 84.2±2.8% (P=.02), respectively, of the baseline with increasing concentrations of niacin (0 to 3.0 mmol/L). Specific ¹²⁵I-HDL protein uptake measured with a 50-fold excess of unlabeled HDL was reduced to as much as 78.3±4.8% (P=.005) in niacin-treated cells. The uptake of labeled cholesterol esters in HDL was unaffected by niacin. Niacin also effected a similar decrease in HDL protein uptake, but not cholesterol esters, from apoA-I–containing HDL particles isolated by immunoaffinity. The conditioned medium obtained from Hep G2 cells incubated with niacin significantly (P=.002) increased cholesterol efflux from cultured human fibroblasts. These data indicate a novel mechanism whereby niacin selectively decreases hepatic removal of HDL apoA-I but not cholesterol esters, thereby increasing the capacity of retained apoA-I to augment reverse cholesterol transport.