Obesity-associated inflammation is characterized by macrophage infiltration into peripheral tissues, contributing to the progression of prediabetes and type 2 diabetes. 12-lipoxygenase (12-LOX) catalyzes the formation of pro-inflammatory eicosanoids and promotes the migration of macrophages, yet its role in obesity-associated inflammation remains incompletely understood. Furthermore, differences between mouse and human orthologs of 12-LOX have limited efforts to study existing pharmacologic inhibitors of 12-LOX. In this study, we used a human gene replacement mouse model in which the gene encoding mouse 12-LOX (Alox15) is replaced by the human ALOX12 gene. As a model of obesity and dysglycemia, we administered male mice a high-fat diet. We subsequently investigated the effects of VLX-1005, a potent and selective small molecule inhibitor of human 12-LOX. Oral administration of VLX-1005 resulted in improved glucose homeostasis, decreased β-cell dedifferentiation, and reduced macrophage infiltration in islets and adipose tissue. Analysis of the stromal vascular fraction from adipose tissue showed a reduction in myeloid cells and cytokine expression with VLX-1005 treatment, indicating decreased adipose tissue inflammation. In a distinct mouse model in which Alox15 was selectively deleted in myeloid cells, we observed decreased β-cell dedifferentiation and reduced macrophage infiltration in both islets and adipose tissue, suggesting that the effects of VLX-1005 may relate to the inhibition of 12-LOX in macrophages. These findings highlight 12-LOX as a key factor in obesity-associated inflammation and suggest that 12-LOX inhibition could serve as a therapeutic strategy to improve glucose homeostasis and peripheral inflammation in the setting of obesity and type 2 diabetes.