Human milk fortifiers (HMF) are commonly added to human milk to increase caloric density and nutrient delivery to preterm infants. TGFβ2 is present in human milk, has immunoregulatory properties, and is involved in growth, differentiation, and repair of neonatal gut epithelia. The effect of HMF on TGFβ2 concentration is unknown. We sought to measure the concentration of TGFβ2 in whole milk following the addition of a commercially available powdered HMF. Human milk samples (N=23) were collected and stored at -20°C. For analysis, milk samples were thawed and divided into two aliquots. HMF was added at a concentration of 0.0384 g/mL (or one 0.96 g packet per 25 mL) to the 2nd aliquot of each pair. Controls included:(1) HMF at a concentration of 0.0384 g/mL in calibrator diluent (CD) and (2) HMF 0.0384 g/mL in CD with recombinant TGFβ2 at 2000 and 500 pg/mL. The concentration of the 25 kD acid-activated TGFβ2 isoform was measured using an ELISA (R&D Systems). The mean±SD concentration of TGFβ2 in unfortified milk was 5554±5345 (range 143-17640) pg/mL human milk. The mean±SD concentration of TGFβ2 in fortified milk was 7135±4774 (range 1641-17390) pg/mL. The mean relative increase in milk TGFβ2 concentration after the addition of HMF was 3.4 times that of unfortified milk. The mean TGFβ2 concentration in HMF control was 2518 pg/mL. HMF doubled the concentration of TGFTGFβ2 standards. In summary, with the addition of HMF to whole milk samples, TGFβ2 concentration decreased 2-to-10% in 4 samples but increased 1-to- 3174% in 19 samples. It appears that HMF is an additional source of TGFβ2 when added to human milk. What effect this alteration in human milk TGFβ2 concentration has on neonatal immune and gut function remains unknown.