The efficacy of extended-release niacin (niacin ER) on lipoprotein subclasses was evaluated in patients with primary hypercholesterolemia using a proton nuclear magnetic resonance method. Paired plasma samples collected at baseline and after 12 weeks’ treatment with niacin ER 1,000 (n = 21) or 2,000 (n = 20) mg/day or placebo (n = 19) were available for 60 eligible patients from a previous multicenter, randomized, controlled trial. Niacin ER increased high-density lipoprotein (HDL) cholesterol and decreased low-density lipoprotein (LDL) cholesterol and very low-density lipoprotein triglycerides in a dose-dependent manner relative to placebo. Niacin ER increased large HDL particles (H5 and H4, corresponding to the HDL_2ab fraction) without having a net effect on small HDL particles (H3, H2, and H1, corresponding to the HDL_3abc fraction). It also decreased smaller, denser LDL particles (L1 and L2) and increased the larger, more buoyant L3 subclass. The inhibitory effect of niacin ER on very low-density lipoprotein was evident on the larger particles (V6, V5, V4, and V3 subclasses) rather than the smaller ones (V2 and V1). The results show that niacin ER produces a beneficial effect on lipoprotein subclasses, specifically decreasing the more atherogenic small, dense LDL particles and enhancing the cardioprotective large HDL particles.