Glucose stimulated insulin secretion (GSIS) was different in rats chronically fed a sucrose-rich diet (SRD) for 3 or 30 wk. This work proposes possible mechanisms underlying insulin secretion changes from β-cell throughout these feeding periods. In isolated islets of rats fed the SRD or a control diet (CD) we examined: 1- the glucokinase and hexokinase activities and their protein mass expression; 2- pyruvate dehydrogenase activity; 3- uncoupling protein 2 (UCP2) and peroxisome proliferators-activated receptor γ (PPAR γ) protein mass expression. At 3 wk on diet the SRD-fed rats showed: a marked increase in the first peak of GSIS; increased glucokinase protein mass expression without changes in glucokinase and hexokinase activities; increased PPARγ protein mass expression without changes in the UCP2 protein mass expression. No changes in either glucose oxidation and triglyceride content within the β-cell were observed. After 30 wk of feeding, a significant decrease of both glucokinase activity and its protein mass expression was accompanied by altered glucose oxidation, a triglyceride increase within the β-cell and a significant increase of PPARγ and UCP2 protein mass expression. Moreover GSIS depicted an absence of the first peak with an increase in the second phase. Finally, the SRD chronic administration altered GSIS by different mechanisms depending on the time on diet. At an early stage, the increased protein mass expression of the glucokinase and a fatty acid cooperative effect inducing PPARγ expression seem to be the mechanisms involved. At a late stage, glucolipotoxicity appears to be the cellular mechanism contributing to progressive β-cell dysfunction.