Steviol glycosides (stevioside and rebaudioside A) have been reported to have lipid and glucose regulatory potential. The published literature presents conflicting results regarding the impact of hyperglycemia on Fe, Zn, and Cu levels, and almost no data exist on whether supplementary steviol glycosides can affect the status of trace elements in diabetes. This study aimed to evaluate the effect of hyperglycemia and dietary steviol glycosides supplementation on Fe, Zn, and Cu levels and the ratios of these elements in the liver and kidney of diabetic rats. The experiment was conducted on 70 male Wistar rats, of which 60 were fed a high-fat diet for 8 weeks, followed by intraperitoneal streptozotocin injection to induce type 2 diabetes, while 10 healthy controls were fed the AIN-93M diet. Afterward, diabetic rats were allocated into the following 6 high-fat diet-fed experimental groups: untreated, supplemented with metformin, or supplemented with stevioside or rebaudioside A (0.5 or 2.5%) for 5 weeks. After the experiment, internal organs were harvested for mineral analyses. The content of Fe, Zn, and Cu in tissues was determined using the AAS method. It was found that hyperglycemia significantly elevated the liver Zn/Cu ratio, simultaneously decreasing the kidney Fe level, as well as Fe/Zn and Zn/Cu ratios in diabetic rats. Supplementary steviol glycosides tended to normalize the kidney Zn/Cu ratio, while high doses of steviol glycosides tended to normalize the kidney Fe concentration in diabetic rats. The type of glycoside differentiated the kidney Zn level and the Fe/Zn ratio in diabetic rats.