OBJECTIVE - Intestinal lipoprotein production has recently been shown to be increased in insulin resistance, but it is not known whether it is regulated by insulin in humans. Here, we investigated the effect of acute hyperinsulinemia on intestinal (and hepatic) lipoprotein production in six healthy men in the presence and absence of concomitant suppression of plasma free fatty acids (FFAs). RESEARCH DESIGN AND METHODS - Each subject underwent the following three lipoprotein turnover studies, in random order, 4-6 weeks apart: 1) insulin and glucose infusion (euglycemic-hyperinsulinemic clamp) to induce hyperinsulinemia, 2) insulin and glucose infusion plus Intralipid and heparin infusion to prevent the insulin-induced suppression of plasma FFAs, and 3) saline control. RESULTS - VLDL1 and VLDL2-apoB48 and -apoB100 production rates were suppressed by 47-62% by insulin, with no change in clearance. When the decline in FFAs was prevented by concomitant infusion of Intralipid and heparin, the production rates of VLDL1 and VLDL2-apoB48 and -apoB100 were intermediate between insulin and glucose infusion and saline control. CONCLUSIONS - This is the first demonstration in humans that intestinal apoB48-containing lipoprotein production is acutely suppressed by insulin, which may involve insulin's direct effects and insulin-mediated suppression of circulating FFAs.