The importance of elevated levels of fatty acids in the pathogenesis of the deteriorated β-cell function present in type 2 diabetes has been established. Long-term exposure of the β-cell to high levels of fatty acids causes enhanced insulin secretion at low glucose (basal insulin release), while glucose-stimulated insulin secretion (GSIS) is decreased or unchanged. We have previously demonstrated that the spatial configuration of fatty acids ( cis and trans isomers) is of importance for the acute impact on the β-cell function. In this study we aimed to elucidate whether the spatial configuration also influenced β-cell function after long-term exposure. Thus, we compared the effect of 3 days culture of INS-1 cells with cis ( cis C 18:1–11) and trans vaccenic acid ( trans C 18:1–11), as well as oleic ( cis C 18:1–9) and elaidic acid ( trans C 18:1–9), on basal and glucose-stimulated insulin release. All fatty acids tested increased basal insulin release; however, a significantly lower basal insulin release was demonstrated for cells cultured with 0.3 to 0.4 mmol/L trans vaccenic acid compared to equimolar levels of the cis isomer. GSIS was not changed by cis or trans vaccenic acid or by oleic acid, whereas it was stimulated by 0.3 to 0.4 mmol/L elaidic acid. The mechanisms behind the fatty acid-induced changes in the β cells have been linked to changes in glucose and fatty acid oxidation. We demonstrated an increased fatty acid oxidation in β cells after long-term exposure to all of the tested fatty acids. Interestingly, both trans isomers ( trans vaccenic and elaidic acid) induced higher fatty acid oxidation than the cis isomers ( cis vaccenic and oleic acid, respectively). No changes in glucose oxidation were found when INS-1 cells were cultured with either of the fatty acids. The increased fatty acid oxidation was associated with an increased content of carnitine palmitoyltransferase I (CPT-I) mRNA, but no difference in the content of CPT-I mRNA to the different fatty acids was found. Insulin mRNA expression in β cells was not affected by the fatty acids. In conclusion, we have demonstrated that the pathological changes in insulin secretion from INS-1 cells to long-term culture with elevated levels of fatty acids are more pronounced for the cis ( cis vaccenic acid and oleic acid) rather than the trans isomers ( trans vaccenic acid and elaidic acid). We suggest that this, at least in part, may be explained by a lower fatty acid oxidation in cells cultured with the cis compared to the trans fatty acid isomers. Apparently, the difference in fatty acid oxidation was not caused by an increased induction of CPT-I mRNA, nor by changes in glucose oxidation or insulin mRNA in β cells chronically exposed to the fatty acids.