Abstract Objective We aim to determine 3-epi-25-OH-D3 in 155 leftover infant samples (<12months) and its relationship with demographic, anthropometric and biochemical markers. Methods Samples were randomly collected and assessed for 3-epi-25-OH-D3 by HPLC. Biochemical data were recorded, if available, including C-reactive protein, calcium, liver and renal markers. Season of birth, gestational age (pre-term vs full-term), weight and height were recorded for infants <1month. Results 3-Epi-25-OH-D3 was quantified in 97% of the samples. Serum levels of the epimer increased at the end of the pregnancy and decreased over the first year of life. 3-Epi-25-OH-D3 was negatively associated with bilirubin and C-reactive protein but unrelated to gestational age, season, age after delivery, height and weight, and calcemia or renal function. Conclusions Based on the present data, both liver function and acute phase reactants should be considered when measuring 3-epi-25-OH-D3 and assessing vitamin D status in infants.