Objective：This study aims to investigate the synergistic effects of mild elevation of free fatty acid and lipopolysaccharide（LPS）on apoptosis and insulin secretion in rat pancreatic β-cell line INS-1 and its mechanism. Methods：The INS-1 cells were cultured with palmitate（0.125 mmol/L）and different concentrations of LPS（1，10，50 ng/mL）for 24 h alone or together. Apoptosis rate was detected by flow cytometry. Western blot was used to detect the expression of Cleaved caspase-3. Neutral ceramidase（NCDase）activity was investigated by HPLC after INS-1 cells were treated with palmitate and LPS（50 ng/mL） alone or together. Insulin secretion was detected by ELISA. After transfected with recombinant plasmids pEGFP-C3-NCDase，the changes of apoptosis rate and insulin secretion of INS-1 cells stimulated by palmitate and LPS were observed. Results：Compared with the control，palmitate alone or LPS（50 ng/mL）alone had no significant effect on apoptosis or insulin secretion. However，the combination of palmitate and LPS significantly increased the apoptosis rate and up-regulated the expression of Cleaved caspase-3（P < 0.05）. Intracellular insulin content or glucose-stimulated insulin secretion was not altered while basal insulin secretion was significantly inhibited by palmitate and LPS for 24 h（P < 0.01）. In addition，NCDase activity was not affected by stimulation with palmitate or LPS alone while its activity was markedly inhibited by combination of palmitate and LPS（P < 0.01）. NCDase overexpression markedly ameliorated the apoptosis induced by palmitate and LPS in INS-1 cells and increased basal insulin secretion（P < 0.05）. Conclusion：Mild elevation of free fatty acid and LPS could synergistically promote β-cell apoptosis and decrease basal insulin secretion through inhibition of the activity of NCDase，a key enzyme of the sphingolipid metabolism.