Objective To explore whether ghrelin can competitively inhibit glucagon-like peptide 1 (GLP-1) to stimulate the secretion of insulin via cyclic adenosinc monophosphate/protein kinase A (cAMP/PKA) pathway. Methods The pancreatic islets were isolated and purified from five 8-10 weeks old male SD rats, and identified by dithizone (DTZ) and acridine orange (AO)/propidium iodide (PI) staining. Sixty pancreatic islets were selected from each rat and then randomly divided into six groups for different disposals:S₀ group (8.3 mmol/L glucose solution), S₁ group (8.3 mmol/L glucose solution+10 nmol/L GLP-1), S₂ group (8.3 mmol/L glucose solution+10 nmol/L GLP-1+10 nmol/L ghrelin), S₃ group (8.3 mmol/L glucose solution+10 nmol/L GLP-1+10 nmol/L ghrelin+1 μmol/L growth hormone-releasing peptide 6[D-Lys³-GHRP-6], an antagonist of growth hormone secretagogue receptor 1α[GHSR-1α]), S₄ group (8.3 mmol/L glucose solution + 10 nmol/L GLP-1+10 nmol/L ghrelin+5 μmol/L forskolin, an adenylate cyclase activator), and S₅ group (8.3 mmol/L glucose solution + 10 nmol/L GLP-1+10 nmol/L ghrelin + 10 μmol/L 6-Phe-cAMP, a PKA activator); all reagents were added after last reagent treatment for 10 min. Each group had a disposal of three hours totally. ELISA assay was used to detect the concentrations of insulin and cAMP. Results The concentrations of insulin and cAMP in S₁ group were significantly higher than those in S₀ group (all P<0.05); their concentrations in S₂ group were significantly lower than those in S₁ group (both P<0.05). The concentrations of insulin and cAMP in S₃, S₄, and S₅ groups were significantly higher than those in S₂ group (all P<0.05). Conclusion Ghrelin can inhibit the effect of GLP-1 in promoting secretion of insulin, which may be mediated by cAMP/PKA pathway.