The functional role of autocrine insulin signaling remains unclear despite considerable investigation. In the present study, we tested the effects of high and low doses of exogenous insulin on Ca2+ signaling, insulin synthesis and insulin secretion in dispersed human islet cells using a combination of imaging, radioimmunoassay and patch-clamp electrophysiology. Although 200 nM insulin stimulated Ca2+ signals with larger amplitudes, the percentage of responding cells was lower when compared with 0.2 nM insulin. However, both 0.2 nM insulin and 200 nM insulin led to a transient increase in accessible cellular insulin content under conditions that glucose did not. This pool of insulin likely reflected de novo synthesis as it could be blocked by cyclohexamide or actinomycin D. Blocking endogenous autocrine insulin signaling in quiescent beta-cells with the insulin receptor inhibitor HMNPA led to a reduction in insulin synthesis, suggesting some degree of basal activity of this positive feed-forward loop. Unlike exposure to high glucose, acute treatment with insulin did not stimulate robust insulin exocytosis, as estimated by C-peptide release and capacitance measurements from single beta-cells. Together these data provide further evidence that autocrine insulin signaling can regulate the function of human pancreatic beta-cells. Our findings suggest autocrine insulin signaling directly controls insulin protein levels, but not exocytosis, in beta-cells and demonstrate the functional specificity of insulin signaling and glucose signaling in human islet cells.