Obesity is defined as increased adiposity, which leads to metabolic disease. The growth of adipose tissue depends on its capacity to expand through hyperplasia or hypertrophy, in order to buffer energy surplus. Also, during the establishment of obesity, adipose tissue expansion reflects adipose lipid metabolism (lipogenesis and/or lipolysis). It is well known that dietary factors can modify lipid metabolism promoting or preventing the development of metabolic abnormalities that concur with obesity. Trans-palmitoleic acid (TP), a biomarker of dairy consumption, has been associated with reduced adiposity in clinical studies. Thus, we aimed to evaluate the effect of TP over adiposity and lipid metabolism-related genes in a rodent model of diet-induced obesity (DIO). To fulfil this aim, we fed C57BL/6 mice with a Control or a High-Fat diet, added with or without TP (3 g/kg diet), during 11 weeks. Body weight and food intake were monitored, fat pads were weighted, histology of visceral adipose tissue was analysed and lipid metabolism-related gene expression was explored by qPCR. Results show that TP consumption prevented weight gain induced by high-fat diet, reduced visceral adipose tissue weight and adipocyte size, while increasing the expression of lipolytic molecules. In conclusion, we show for the first time that TP influences adipose tissue metabolism, specifically lipolysis, resulting in decreased adiposity and reduced adipocyte size in a DIO mice model.