Aims: The aim of this study was to examine the chronic effects of different non-esterified fatty acids (NEFA) on insulin secretion by pancreatic islets of normal Wistar rats in vitro. Methods: Pancreatic islets were isolated from normal Wistar rats, and were incubated with 0.2, 0.4, or 0.8 mmol/L palmitate (Cl 6:0), stearate (Cl 8:0), oleate (Cl 8: 1), or linoleate (Cl 8:2) for 24 h, then the insulin secretion and pyruvate dehydrogenase (PDH) activity were examined. Results: Neither islet insulin content nor islet DNA content differed among islets incubated with each kind of NEFA. Compared with control, linoleate significantly inhibited glucose-stimulated insulin secretion (GSIS) and PDH activity at each concentration (p < 0.05), while others inhibited GSIS and PDH activity significantly only at 0.4 and 0.8 mmol/L (p < 0.05). There was no significant difference in GSIS and PDH activity among islets pretreated by palmitate, stearate, and oleate at the same concentration (p > 0.05). However, linoleate decreased GSIS more than others at the same concentration (p < 0.05), while linoleate (0.4 or 0.8 mmol/L) inhibited PDH activity more than others at the same concentration (p < 0.05). Conclusions: Elevation of palmitate, stearate, oleate or linoleate decreases the beta-cell secretory response to glucose, through inhibiting PDH activity. Linoleate exerts more negative effect on GSIS than other NEFA.