D-Lactate is produced by the intestinal biota and later absorbed into circulation. Some patients with cystic fibrosis (CF) develop exocrine pancreatic insufficiency that may disturb the gut microbiome and enhance the production of D-lactate. However, this concept has not been studied yet. The aim of the study was to assess D-lactate concentration in relation to the occurrence of clinical features, activity of CF, and diet composition in paediatric patients. Patients and Method. Serum concentrations of D-lactate were measured in 38 CF patients (19 girls and 19 boys) from 6 months to 18 years of age. The analysis included age, sex, clinical symptoms, diet (the variety and calorie needs), the laboratory tests for pancreatic efficiency (serum levels of albumin and glucose, faecal elastase activity, and faecal fat index) and faecal calprotectin (the marker of intestinal inflammation), and parameters of liver damage and of cholestasis (the activity of aminotransferases, γ-glutamyltransferase, level of bilirubin, and international normalized ratio). Results. The median level of D-lactate was 0.86 μg/ml (1Q–3Q: 0.48–2.03) and correlated with the CF severity in the Schwachman-Kulczycki score, parameters of pancreatic insufficiency, and the presence of intestinal inflammation. An increased level of D-lactate was observed in the subgroup with pancreas insufficiency (1.05 versus 0.73; p < 0.05), parallel with an elevated level of calprotectin (0.948 versus 0.755; p = 0.08). There was no relationship between energy consumption and diet composition and serum D-lactates. Conclusion. Serum D-lactate concentration in CF patients is a promising new marker of exocrine pancreatic insufficiency probably related to intestinal flora dysbiosis/overgrowth.