FOXP3⁺ regulatory T cells (Treg) suppress innate and adaptive immune responses and are critical for intestinal immune homeostasis. Our objective was to define the postnatal developmental regulation of Treg in relationship to other T cells in the human intestinal tract. We analyzed 41 small and 18 large intestinal paraffin-embedded tissue samples from preterm and term infants with and without necrotizing enterocolitis (NEC) for the presence of CD3⁺, CD4⁺, CD8⁺, and FOXP3⁺ cells by immunohistochemistry. We compared labeled cells against age, gestational age (GA), or (corrected) postmenstrual age (PMA). The GA ranged from 23 to 40 weeks, with a mean of 32 (standard deviation, 4.7) weeks. Independent of age, GA, or PMA, the numbers of CD4⁺ cells were higher in the small intestine compared to the large intestine (P = 0.046), except in patients with NEC. FOXP3⁺ cells could be detected as early as 23 weeks in GA in both large and small bowel, and similar quantities were detected at the highest GA examined (40 weeks). We saw no statistically significant effect of GA, age, or PMA on total number of FOXP3⁺ cells or by comparing FOXP3⁺ to CD4⁺ or FOXP3⁺ to CD8⁺ ratios, indicating intact ontogeny of Treg in intestinal tissue early in gestation. Human infants exhibit presence of mucosal FOXP3⁺ cells in the small and large intestinal mucosa at birth and as early as 23 weeks GA. The frequency of FOXP3⁺ cells and the ratios of FOXP3⁺ to CD4⁺ or CD8⁺ cells do not change with increasing intrauterine development or postnatal age.