Type 1 diabetes mellitus (T1DM) is associated with neurocognitive dysfunction and astrogliosis. Physical exercise prevents cognitive impairments and induces important brain modifications. The aim of our study was to investigate the effect of treadmill exercise on spatial memory and astrocytic function in the hippocampus of a T1DM model. Fifty-seven Wistar rats were divided into four groups: trained control (TC) (n = 15), non-trained control (NTC) (n = 13), trained diabetic (TD) (n = 14) and non-trained diabetic (NTD) (n = 15). One month after streptozotocin-induced diabetes, exercise groups were submitted to 5 weeks of physical training, and then, all groups were assessed in the novel object-placement recognition task. Locomotor activity was analyzed in the open field apparatus using Any-maze software. The expression of glial fibrillary acidic protein (GFAP) and S100B in hippocampus and cerebrospinal fluid were measured using ELISA assay, and hippocampal GFAP immunoreactivity was evaluated by means of immunohistochemistry and optical densitometry. The results showed that physical exercise prevents and/or reverts spatial memory impairments observed in NTD animals (P < 0.01). Decreased locomotor activity was observed in both the NTD and TD groups when compared with controls (P < 0.05). ELISA and immunohistochemistry analyzes showed there was a reduction in GFAP levels in the hippocampus of NTD animals, which was not found in TD group. ELISA also showed an increase in S100B levels in the cerebrospinal fluid from the NTD group (P < 0.01) and no such increase was found in the TD group. Our findings indicate that physical exercise prevents and/or reverts the cognitive deficits and astroglial alterations induced by T1DM.