Both mitochondrial dysfunction and alterations in mitochondrial DNA (mtDNA) are implicated in type 2 diabetes mellitus and insulin resistance. Evidence also suggests that metabolism of S-adenosyl-L-methionine (SAM), the universal methyl donor for biological methylation, is associated with mitochondrial dysfunction and insulin resistance. We investigated the effect of SAM on mtDNA density and insulin sensitivity using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of type 2 diabetes mellitus and insulin resistance. To determine the short-term effect on mtDNA density, SAM (15 mg.kg-1.d-1) was administered intraperitoneally for 7 d to 6 male, 57-wk-old OLETF rats and 6 Long-Evans Tokushima Otsuka (LETO) rats of the same age as a nondiabetic control. To determine the long-term effect, the same dose of SAM was administered daily to 5 male, 6-wk-old OLETF rats until the age of 25 wk; 7 control OLETF rats received vehicle and 7 LETO rats were untreated. Skeletal muscle mtDNA density was measured by either competitive or multiplex PCR and insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. SAM treatment for 1 wk increased skeletal muscle mtDNA density of both OLETF and LETO rats. The long-term SAM treatment significantly reduced body weight gain as well as increased skeletal muscle mtDNA density and whole body insulin sensitivity in OLETF rats compared with their vehicle-treated controls. Furthermore, in all 3 groups, skeletal muscle mtDNA density correlated with insulin sensitivity (r=0.752, P<0.001). In conclusion, SAM treatment increased mtDNA density in the skeletal muscle, improved whole body insulin sensitivity, and prevented body weight gain in OLETF rats.