As data on food contamination with the mycotoxin citrinin (CIT) are scarce, a recently developed method for biomarker analysis (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013) was applied to investigate CIT exposure of German adults. CIT and its human metabolite dihydrocitrinone (HO-CIT) were determined in urine samples from a group of 50 healthy adults (n = 27 females and n = 23 males). After cleanup by immunoaffinity (CitriTest®) columns, extracts were analyzed by LC-MS/MS. The mycotoxin and its major metabolite HO-CIT were detected in 82 and 84 % of all urine samples, at concentrations ranging from 0.02 (limit of detection, LOD) to 0.08 ng/mL for CIT, and 0.05 (LOD) to 0.51 ng/mL for HO-CIT. Median urine analyte levels in the cohort were 0.03 (CIT) and 0.06 ng/mL (OH-CIT) or adjusted to creatinine 20.2 ng/g crea (CIT) and 60.9 ng/g crea (HO-CIT), respectively. Except for higher urinary CIT levels in males, differences between subgroups were not significant. This first biomarker analysis indicates widespread and variable exposure to CIT in German adults, and conversion of ingested mycotoxin to its less toxic metabolite HO-CIT, which may serve as biomarker of exposure in addition to the parent compound.