Vaccenic acid (VA, trans11-18:1) and cis-9, trans11-conjugated linoleic acid (also known as rumenic acid; RA) have received widespread attention as “beneficial” trans fatty acids due to their putative health benefits, including anti-diabetic properties. Feeding oilseeds (e.g., sunflower seeds or flaxseeds) to cattle given forage-based diets has been shown to enrich their meat and milk fat with VA and RA. However, there is a shortage of studies evaluating the health effects of the resulting enriched fat. The objective of this research was to determine the effects of beef fat enriched with VA and RA on parameters related to glucose homeostasis and liver health in diet-induced obese (DIO) mice. Thirty-six 8-week-old male C57BL/6 mice were divided into nine cages of similar average body weight (four mice per cage). Each cage was randomly assigned for 18 weeks to either a control low-fat diet (CLF; 10% total calories from fat), a control high-fat diet containing lard (CHF), or an enriched-high fat diet containing beef fat enriched with VA and RA (EHF). The enriched beef fat used in EHF was collected from steers fed a diet containing 75% hay and 25% flaxseed-based concentrate. The CHF and EHF were isonitrogenous and isocaloric, providing 45% of total calories from fat. Body weight and food intake were measured weekly throughout the study. Blood triglycerides, total cholesterol, glucose, and insulin concentrations were determined at week 15 using commercial kits. Plasma aspartate aminotransferase (AST) and alanine transaminase (ALT) were analyzed using plasma from week 15. Glucose and insulin tolerance tests (GTT and ITT) were performed on weeks 16 and 17, respectively. After 18 weeks on the dietary treatments, mice were euthanized by cervical dislocation and liver tissue collected to assess liver morphology and histology. Liver triglyceride content was measured with a colorimetric diagnostic kit. The fatty acid composition of liver was determined using gas chromatography (GC) and hepatic expression of inflammation markers was determined using qPCR. Data were analysed using the mixed models procedure of SAS and differences between means were considered significant at P < 0.05 using the Tukey−Kramer multiple comparison test. Compared with CLF, feeding either CHF or EHF resulted in negative metabolic outcomes associated with high-fat diets, including adiposity, impaired insulin sensitivity and glucose tolerance, and hepatic steatosis. However, the EHF diet induced a higher (P < 0.05) liver weight, and hepatic triglyceride content and resulted in a lower hepatic n-3 and n-6 Polyunsaturated fatty acids (PUFA) content compared with the CHF group. In parallel with steatosis, plasma levels of ALT and hepatic expression of MCP-1 and F4/80 were highest in the EHF group. In addition, the area under the curve for the GTT in EHF mice was higher (P < 0.05) than in the CHF group. Collectively, the findings from this research suggest that feeding beef fat enriched with VA and RA (i.e. EHF) adversely impact glucose tolerance and liver steatosis in diet-induced obese mice. Additional studies are needed to determine if the adverse effects of EHF on glucose tolerance and hepatic lipid accumulation are related to VA and RA or due to other natural trans-fatty acids present in EHF.