Obesity arises from a complex interplay of genetic and environmental factors. Even among individuals with the same genetic predisposition, diet-induced obesity (DIO) exhibits varying degrees of susceptibility, which are categorized as DIO and diet-induced obesity resistance (DR). The hypothalamus plays a pivotal role in regulating energy homeostasis. This study performed a comparative hypothalamic proteomic analysis in DIO and DR rats to identify differentially expressed proteins (DEPs) associated with alterations in body weight. Male Sprague Dawley rats were fed either a standard chow diet or a high-fat diet for 12 weeks. DIO rats exhibited the most rapid weight gain compared to both the control and DR rats. Despite consuming similar caloric intake, DR rats exhibited less weight gain relative to DIO rats. Proteomic analysis revealed 31 DEPs in the hypothalamus of DR rats compared to DIO rats (with a false discovery rate (FDR) <
1%). Notably, 14 proteins were upregulated and 17 proteins were downregulated in DR rats. Gene ontology analysis revealed an enrichment of ion-binding proteins, such as those binding to Fe