Treg cells are crucial to prevent immune dysregulation, but little is known about the frequency of these cells in neonates, particularly in very/moderate and late preterm newborns studied as separate groups. The CD4+CD25hiCD127loFOXP3+Treg population was phenotypically characterized to assess maturation markers and gut-homing integrins by flow cytometry in the cord blood of healthy preterm newborns born at 30–336/7 gestation weeks (Group 1), at 34–366/7 gestation weeks (Group 2) and term newborns born at 37–41 gestation weeks (Group 3), compared to healthy adults. An inverse correlation of the Treg percentage and gestational age was found, with significantly higher frequencies in Group 1 compared to Groups 2 and 3 and in Group 2 compared to Group 3, and significantly higher Treg frequencies and numbers in the neonates compared to the adults. All of the newborns exhibited increased Treg frequencies with a naive phenotype compared to adults. Cytotoxic T-lymphocyte-associated protein 4 CTLA-4 expression in the naive Treg was decreased in both preterm groups compared with those from term newborns and adults, and in the memory Treg from Group 1 compared with the other groups. The frequencies of Treg expressing α4β7 and α4β1 integrins were higher in both preterm groups, but significantly different only in Group 1, when compared with those from the term newborns and the adults. In conclusion, although a high frequency of Treg is present in newborns, an immature phenotype with a higher expression of CD45RA and α4β7/α4β1 and a lower expression of CTLA-4 is found, particularly in the very preterm group.