Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.