It is currently unknown why obesity leads in some patients to prediabetes and metabolic syndrome. Microinflammation potentially caused by extracellular DNA is supposed to be involved. The aim of this cross-sectional study in healthy mice was to analyze the association between plasma extracellular DNA and glucose metabolism. Fasting glycemia and insulin were measured in healthy adult female mice that subsequently underwent an oral glucose tolerance test. Indices of glucose metabolism and insulin sensitivity were calculated. DNA was isolated from plasma and quantified fluorometrically. Deoxyribonuclease (DNase) activity of plasma was measured using the single radial enzyme diffusion method. Fasting glycemia correlated negatively with both, extracellular DNA and DNase (r = -0.44 and r = -0.32, respectively). DNase was associated positively with the incremental area under curve (r = 0.35), while extracellular DNA correlated negatively with total area under curve of glycemia during oral glucose tolerance test (r = -0.34). Measures of insulin sensitivity were found to be associated with neither extracellular DNA, nor DNase. The hypothesis of an association of low DNase with increased fasting glucose was partially proved. Surprisingly, low extracellular DNA is associated with higher fasting glucose and lower glucose tolerance in mice. As novel therapeutic targets for prediabetes and metabolic syndrome are highly needed, this study provides novel unexpected associations within the limitations of the focus on physiological variability as it was conducted on healthy mice. The causality of these associations should be proved in further interventional experiments.