Cardiac autonomic dysfunction and cardiac microvascular dysfunction are diabetic complications associated with increased mortality, but the association between these has been difficult to assess. We applied new and sensitive methods to assess this in patients with type 2 diabetes mellitus (T2DM). In a cross-sectional design, coronary flow reserve (CFR) assessed by cardiac (82)Rb-positron emission tomography/computed tomography, cardiac autonomic reflex tests, and heart rate variability indices were performed in 55 patients with T2DM, without cardiovascular disease, and in 28 control subjects. Cardiac (123)I-metaiodobenzylguanidine scintigraphy was conducted in a subgroup of 29 patients and 14 control subjects and evaluated as the late heart-to-mediastinum ratio and washout rate. Impaired function of all the cardiac autonomic measures (except the washout rate) was associated with reduced CFR. A heart rate variability index, reflecting sympathetic and parasympathetic function (low-frequency power), and the late heart-to-mediastinum ratio, reflecting the function of adrenergic receptors and sympathetic activity, were positively correlated with CFR after adjustment for age and heart rate. The late heart-to- mediastinum ratio remained correlated with CFR after further adjustment. In patients with T2DM without cardiovascular disease, we demonstrate an independent association between cardiac autonomic function and CFR. We suggest that a reduced cardiac autonomic function and damage to the adrenergic receptors may contribute to the development of cardiac microvascular dysfunction.