The risk of cognitive impairment is markedly elevated in patients with type 2 diabetes mellitus (T2DM). While exercise has been shown to mitigate cognitive deficits associated with diabetes, the underlying mechanisms remain poorly understood. Recent studies suggest that exercise can modulate the composition of the gut microbiota, which, in turn, may influence the central nervous system via the microbiota-gut-brain axis. However, the specific role of gut microbiota in mediating exercise-induced improvements in cognitive function in T2DM remains unclear. In this study, we aimed to investigate whether exercise can alleviate cognitive impairment in T2DM mice by modulating the intestinal microbiota, and to elucidate the mechanisms underlying this effect. This study was conducted using male C57BL/6J mice. Mice fed a normal diet were assigned to the non-diabetic control group (NC), while those fed a high-fat diet were intraperitoneally injected with streptozotocin (STZ) and subsequently divided into the diabetic control group (DM), an exercise group (DM-EXE), and a fecal microbiota transplantation group (DM-FMT). The DM-EXE group underwent treadmill exercise for 8 weeks. During this period, the DM-FMT group received fecal microbiota transplants from the DM-EXE group for 2 consecutive days per week. Following the 8-week intervention, stool samples were collected for 16S rDNA high-throughput sequencing. The fear conditioning test was performed to assess cognitive function. Intestinal mucosa samples were collected to evaluate the expression of intestinal tight junction proteins. Additionally, the expression levels of synaptic proteins, glucose transporters, neurotrophic factors, and inflammatory markers were measured in the hippocampus. Our findings demonstrate that T2DM mice exhibit impaired cognitive function and significant alterations in their gut microbiota compared to non-diabetic controls. Exercise partially reversed these changes in the intestinal microbiota and alleviated cognitive impairment in T2DM mice. Additionally, transplantation of intestinal microbiota from exercised mice improved cognitive function in T2DM mice. Aerobic exercise may mitigate cognitive impairment in T2DM mice by modulating the gut microbiota. The underlying mechanisms appear to involve enhanced neural synaptic plasticity, reduced neuroinflammation, and improved neuronal glucose metabolism.