The effect of acute hyperinsulinemia on plasma cholesteryl ester (CE) transfer protein (CETP) activity was determined in 11 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 10 healthy subjects. Plasma CETP activity was reduced significantly in NIDDM patients (−37 ± 59 nmol/mL/h, P < .05) but not in healthy subjects (−7 ± 37 nmol/mL/h) during insulin infusion. Saline infusion did not alter plasma CETP activity significantly. The change in plasma CETP activity was correlated significantly with the baseline plasma triglyceride (TG) concentration ( r = −.523, n = 21, P = .01) and marginally with the concomitant decrease in these levels with acute hyperinsulinemia ( r = .413, n = 21, P = .06) in NIDDM patients and healthy subjects combined. These data indicate that acute hyperinsulinemia reduces plasma CETP activity and probably plasma CETP concentration in NIDDM patients, and suggest coordinated regulation of CETP levels and TG metabolism by insulin.