Recent obesity research has reported differences in intestinal microbiota between obese ob/ob mice and lean wild type mice suggesting a potential link between bacterial populations and metabolic health. Sterculic oil (SO) is a natural inhibitor of stearoyl-CoA desaturase-1, a potential therapeutic target for the treatment/attenuation of obesity and insulin resistance.The aim of this study was to investigate the response of the obese and lean gut microbiota to a SO supplemented diet. Five-week old male lean (WT) and obese (ob/ob) mice were fed either a control AIN-93G diet or an AIN-93G diet containing 0.5% SO for 9 weeks Barcoded pyrosequencing of the V4-V5 region of the 16S rRNA gene was employed to characterize cecal microbiota. Intestinal microbiota from WT mice fed a control diet were more diverse, although there were no significant differences in the proportions of major phyla from WT and ob/ob mice. Irrespective of genotype, SO was negatively correlated with the relative abundances of Actinobacteria, Bacilli and Erysipelotrichi. This shift was not associated with a change in body weight, however a statistically significant improvement in glucose tolerance and insulin sensitivity was observed in the ob/ob mice fed SO. In our study, diet had a greater impact on gut microbiota as compared to genotype and these changes were associated with an improved metabolic health.