Metabolic impairments associated with type 2 diabetes, including insulin resistance and loss of glycaemic control, disproportionately impact the elderly. Lifestyle interventions, such as manipulation of dietary fat quality (i.e. fatty acid (FA) composition), have been shown to favourably modulate metabolic health. Yet, whether or not chronic consumption of beneficial FAs can protect against metabolic derangements and disease risk during ageing is not well defined. We sought to evaluate whether long-term dietary supplementation of fish-, dairy- or echium-derived FAs to the average FA profile in a U.S. American diet may offset metabolic impairments in males and females during ageing. One-month-old CD-1® mice were fed isoenergetic, high-fat (40 %) diets with the fat content composed of either 100 % control fat blend (CO) or 70 % CO with 30 % fish oil, dairy fat or echium oil for 13 months. Every 3 months, parameters of glucose homoeostasis were evaluated via glucose and insulin tolerance tests. Glucose tolerance improved in males consuming a diet supplemented with fish oil or echium oil as ageing progressed, but not in females. Yet, females were more metabolically protected than males regardless of age. Additionally, Spearman correlations were performed between indices of glucose homoeostasis and previously reported measurements of diet-derived FA content in tissues and colonic bacterial composition, which also revealed sex-specific associations. This study provides evidence that long-term dietary fat quality influences risk factors of metabolic diseases during ageing in a sex-dependent manner; thus, sex is a critical factor to be considered in future dietary strategies to mitigate type 2 diabetes risk.