Hyperglycemia is linked to a higher risk of diabetes, epilepsy, and seizures, which contribute to increased mortality. Previous research has shown that hyperglycemia impairs the function of the polyamine-dependent Kir4.1 channels in cultured astrocytes, and a similar effect was observed in male astrocytes from diabetic (db/db) mice. However, whether there are sex differences in this effect remains unclear. This study aims to compare Kir4.1 channel function in 10–12 week old brains of female db/db mice with that in males and non-diabetic heterozygous (db/+). We performed mRNA analyses, Western blotting (WB), glucose level measurements, and patch-clamp recordings from hippocampal astrocytes in brain slices to assess Kir4.1 channel activity in db/db and db/+ mice of both sexes. Our results showed increased glucose levels in diabetic db/db mice, especially in males, along with (1) reduced synthesis of Kir4.1 channel protein, (2) reduced membrane potential, (3) decreased Kir currents, and (4) compromised potassium uptake. Female diabetic astrocytes exhibited significantly lower barium-blocked Kir4.1 currents compared to diabetic males. Additionally, barium-insensitive currents (leakage currents) were upregulated in db/db mice, likely as a compensatory response to hyperglycemia. In conclusion, diabetes impairs astrocyte function by downregulating Kir4.1 channels, with a more pronounced effect in male diabetic mice. This impairment may increase seizure risk by affecting the ability of astrocytes to maintain extracellular ion balance.