Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting.We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro.We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis.Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.