Background: Only few studies have quantitated the frequencies of immune cells in the small bowel mucosa and submucosa during gestation. The aims of this study were to describe the frequencies of T and B cells, eosinophils and mast cells in the normal small bowel mucosa and submucosa (NSB) in relation to gestational age (GA) and in the uninvolved small bowel (USB) of premature newborns with necrotizing enterocolitis (NEC). Methods: We obtained 36 NSB specimens (GA 12–41 weeks) and 8 NEC-USB specimens (GA 24–32 weeks) from autopsies and surgeries and performed immunostaining for CD3, CD79a, BMK-13 and tryptase as well as the histochemical stains giemsa and toluidine blue. Qualitative histological evaluation and two different quantitative cell-samplings were performed using digital imaging analysis with both TissuemorphDP ^® and newCAST® software. Linear regression analysis was performed with cell frequency as the dependent variable and GA and USB as the independent variables. Results: In the NSB specimens, we found significant linear correlations between cell frequencies and GA for all examined cell types, though B cell frequencies reached a plateau midway through gestation. In the USB cases, submucosal mast cell frequencies were higher than in the NSB specimens, while T cell frequencies were lower. In USB of NEC patients, we found a significant increase of mast cells and a significant decrease of T cells compared to NSB. Conclusion: Throughout gestation, we found an increase of all examined immune cell types in the normal small bowel, while the number of B cells came to a standstill at midway. Future studies should examine subtypes of T cells and also include histiocytes. A larger amount of small bowel specimens, covering the full gestational age, would be of great value.