Higher levels of the adipocyte-specific hormone adiponectin have been linked to increased high-density lipoprotein (HDL) and lower insulin resistance. This study was conducted to determine the influence of macronutrient intake on adiponectin levels. One hundred and sixty-four pre- and stage-1 hypertensive adults participated in the Optimal Macro-Nutrient Intake Heart (OMNI-Heart) trial, a crossover feeding study originally testing the effects of macronutrients on blood pressure. Participants underwent three 6-week feeding periods: one rich in carbohydrates (CARB), one rich in monounsaturated fat (MUFA), and one rich in protein (PROT), while maintaining body weight. Their median plasma high molecular weight (HMW) and total adiponectin levels were 2.3 and 8.2 microg/ml, respectively, resulting in an average of 27% HMW adiponectin. Both HMW and total adiponectin levels decreased after baseline while the percent HMW adiponectin remained unchanged. Between diets, the MUFA diet maintained a higher level of both HMW and total adiponectin levels than either the CARB (HMW: +6.8%, P = 0.02; total: +4.5%, P = 0.001) or PROT (HMW: +8.4%, P = 0.003; total: +5.6%, P < 0.001) diets. Changes in total adiponectin levels were positively correlated to changes in HDL cholesterol irrespective of diets (Spearman r = 0.22-0.40). No correlation was found between changes in lipids, blood pressure, or insulin resistance by the homeostasis model assessment (HOMAIR). Macronutrient intake has effects on HMW and total adiponectin levels independent of weight loss. A diet rich in MUFA was associated with higher levels of total and HMW adiponectin in comparison to a carbohydrate- or protein-rich diet. Effects seen in adiponectin paralleled those found with HDL cholesterol.