BACKGROUND Nuclear receptor subfamily 4 group A member 1 (Nr4a1) has been increasingly investigated in association with type 2 diabetes mellitus (T2DM). This study aimed to explore its efficacy with liver kinase B1 (LKB1) and potential signaling pathways in T2DM. MATERIAL AND METHODS A T2DM model in rats was established by high-fat diet and injection of 30 mg/kg streptozotocin. The ectopic expression of Nr4a1 or in combination with LKB1 was performed in T2DM rats to probe their effects on T2DM. Then, the weight and indicators of blood lipid and blood glucose in normal rats and T2DM rats were measured. The volume change of adipocytes and the size of lipid droplets in white adipose tissue (WAT) were observed by hematoxylin-eosin staining and oil red O staining, respectively. We also measured levels of Nr4a1, LKB1, and adenosine monophosphate-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/Nuclear factor-kappa B (NF-kappaB) axis-related proteins. RESULTS In T2DM rats, Nr4a1 was highly expressed, and body weight, blood lipid and blood glucose were increased, and the volume of adipocytes and the size of lipid droplets in WAT were increased, which were all reversed by low expression of Nr4a1. After treatment with Nr4a1 and LKB1 together, T2DM rats showed decreased levels of blood lipid, blood glucose, and reduced volume of adipocytes and lipid droplet size in WAT, with activated AMPK/SIRT1 signaling pathway and inhibited NF-kappaB. CONCLUSIONS Our results highlight that interaction of Nr4a1 and LKB1 can mitigate T2DM by activating the AMPK/SIRT1 signaling pathway and inhibiting NF-kappaB activation. This may offer new insight for T2DM treatment.