The orexigenic hormone ghrelin is important in diabetes because it has an inhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin^−/−:ob/ob) mice increases insulin secretion and improves hyperglycemia. The physiologically relevant ghrelin receptor is the growth hormone secretagogue receptor (GHS-R), and GHS-R antagonists are thought to be an effective strategy for treating diabetes. However, since some of ghrelin's effects are independent of GHS-R, we have utilized genetic approaches to determine whether ghrelin's effect on insulin secretion is mediated through GHS-R and whether GHS-R antagonism indeed inhibits insulin secretion. We investigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr^−/−:ob/ob). Ghsr ablation did not rescue the hyperphagia, obesity, or insulin resistance of ob/ob mice. Surprisingly, Ghsr ablation worsened the hyperglycemia, decreased insulin, and impaired glucose tolerance. Consistently, Ghsr ablation in ob/ob mice upregulated negative β-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive β-cell regulators (such as HIF-1α, FGF-21, and PDX-1) in whole pancreas; this suggests that Ghsr ablation impairs pancreatic β-cell function in leptin deficiency. Of note, Ghsr ablation in ob/ob mice did not affect the islet size; the average islet size of Ghsr^−/−:ob/ob mice is similar to that of ob/ob mice. In summary, because Ghsr ablation in leptin deficiency impairs insulin secretion and worsens hyperglycemia, this suggests that GHS-R antagonists may actually aggravate diabetes under certain conditions. The paradoxical effects of ghrelin ablation and Ghsr ablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.