Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic β-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by β-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic β-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca²⁺ chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca²⁺ and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.