Low HDL is a risk factor for the development of type 2 diabetes. Hepatic ABCA1 is the rate-limiting protein in HDL biogenesis, and mice lacking hepatic ABCA1 (ABCA1(-l/-l)) have very low plasma HDL concentrations. To investigate the role of hepatic ABCA1 in glucose tolerance and β-cell function, we used ABCA1(-l/-l) mice, which showed impaired glucose tolerance without changes in insulin sensitivity. Insulin secretion was reduced following glucose gavage. Ex vivo, glucose stimulated insulin secretion from β-cells from wild-type (WT) and ABCA1(-l/-l) mice was similar. Insulin secretion was, however, reduced upon addition of ABCA1(-l/-l) serum to the medium compared with WT serum, whereas islets lacking β-cell ABCA1 were not affected differently by ABCA1(-l/-l) or WT serum. After high-fat feeding, WT and ABCA1(-l/-l) mice showed no difference in glucose tolerance or insulin secretion, and serum from ABCA1(-l/-l) and WT mice fed a high-fat diet did not affect insulin secretion differently. We conclude that hepatic ABCA1 improves glucose tolerance by improving β-cell function through both HDL production and interaction with β-cell ABCA1. The beneficial effect of hepatic ABCA1 is decreased under metabolic stress. Increasing hepatic ABCA1 may represent a novel therapeutic strategy for improving glucose homeostasis in diabetes.